Sunday, May 1, 2011

Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo

text
Original Article

Acta Pharmacologica Sinica (2010) 31: 821–830; doi: 10.1038/aps.2010.67; published online 28 June 2010
Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo

Chen Zhang1, Kun-zheng Wang1, Hui Qiang1, Yi-lun Tang1, Qian Li2, Miao Li2 and Xiao-qian Dang1

1. 1Department of Orthopedic Surgery, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China
2. 2Department of Ultrasound, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China

Correspondence: Xiao-qian Dang, E-mail dang_xiaoqian@sohu.com

Received 22 February 2010; Accepted 6 May 2010; Published online 28 June 2010.
Top of page
Abstract
Aim:


To investigate the therapeutic potential of adeno-associated virus (AAV)-mediated expression of vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP).
Methods:


Four experimental groups were administered the following AAV vector constructs: rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7 (AAV-VEGF/BMP), rAAV-hVEGF165-GFP (AAV-VEGF), rAAV-hBMP-7-GFP (AAV-BMP), and rAAV-IRES-GFP (AAV-GFP). VEGF165 and BMP-7 gene expression was detected using RT-PCR. The VEGF165 and BMP-7 protein expression was determined by Western blotting and ELISA. The rabbit ischemic hind limb model was adopted and rAAV was administered intramuscularly into the ischemic limb.
Results:


Rabbit bone marrow-derived mesenchymal stem cells (BMSCs) were cultured and infected with the four viral vectors. The expression of GFP increased from the 7th day of infection and could be detected on the 28th day post-infection. In the AAV-VEGF/BMP group, the levels of VEGF165 and BMP-7 increased with prolonged infection time. The VEGF165 and BMP-7 secreted from BMSCs in the AAV-VEGF/BMP group enhanced HUVEC tube formation and resulted in a stronger osteogenic ability, respectively. In rabbit ischemic hind limb model, GFP expression increased from the 4th week and could be detected at 8 weeks post-injection. The rAAV vector had superior gene expressing activity. Eight weeks after gene transfer, the mean blood flow was significantly higher in the AAV-VEGF/BMP group. Orthotopic ossification was radiographically evident, and capillary growth and calcium deposits were obvious in this group.
Conclusion:


AAV-mediated VEGF and BMP gene transfer stimulates angiogenesis and bone regeneration and may be a new therapeutic technique for the treatment of avascular necrosis of the femoral head (ANFH).
Keywords:

Thursday, April 28, 2011

The Biotechnology Industry By Richard Graydon, M.D.



The primary goal of the biotechnology industry is to invent new biologically-active substances, for the treatment of disease, the pharmaceutical industry, and the management of agriculture.

The U.S. is the world leader in biotechnology, currently providing employment for more than 300,000 people in over 6,000 U.S. biotech companies, with an estimated market capitalization of approximately USD 281 billion in 2008. The biotechnology industry has more than tripled in size since 2000, with revenues increasing from USD 25 billion in 2000 to more than USD 80 billion in 2008. In 2007, Amgen, Inc., the world's largest biotechnology company, achieved total sales of USD 14.7 billion. Expectations are that the biotechnology market will increase to more than USD 130 billion by 2011.

Geographically, the U.S. accounts for 65% of the biotechnology market, Europe 23%, Canada 7%, Australia 3%, and the rest of the world, 2%.

Wednesday, April 27, 2011

Motivated research

Motivated research

Antoine Danchin
Three years ago, a senior politician attended his country's Annual Congress for the Advancement of Science to give the introductory lecture. He asked the attending scientists to make science and research more attractive to young students and the general public, and asked his countrymen to support scientists to address the urgent challenges of global climate change, energy needs and dwindling water resources.

if you student or college in university  you must have

Motivated research for case ctuy, discustion and sharing

Press release Polaris Software Lab Limited (POLARIS.NS)(532254.BO)


ts release from http://www.businesswireindia.com if you share release gaian or new release you can email in here
Source: Polaris Software Lab Limited (POLARIS.NS)(532254.BO)
Wednesday, April 27, 2011 11:49 AM IST (06:19 AM GMT)
Editors: General: Economy; Business: Business services, Defence & security, Financial Analyst, Information technology, Stock exchanges; Technology

Polaris Enters Cloud Computing Space through Strategic Investment in IdenTrustTM
Investment in US-based Global Leader in Trusted Identity Solutions uniquely positions Polaris to drive the most robust digital security infrastructure in the US & European markets

Chennai, Tamil Nadu, India and New York, United States, Wednesday, April 27, 2011 -- (Business Wire India)


Polaris Software Lab Limited (POLS.BO), a leading global Financial Technology Company today announced a strategic investment in IdenTrustTM, a global leader in trusted identity solutions recognized by global financial institutions and one of the premier providers of digital identity authentication services to several key banks, United States federal identity programs as well as supply chain markets. This strategic investment will mark Polaris’ entry into the cloud computing space for Financial Technology solutions.

IdenTrust™ Inc. was founded in 1999 by a group of financial institutions that included Citigroup, Bank of America, Chase, Barclays, HSBC, and Deutsche. As the only bank-developed identity authentication system, IdenTrustTM provides a unique legally and technologically interoperable environment for authenticating and using identities worldwide. IdenTrustTM provides applications that use electronic identities that establish contracts, grant authority, support secure encrypted data and information storage and secure online interactions/transactions.

Monday, April 25, 2011

Beberapa tips memilih emas

Beberapa tips pemilihan emas:
  1. Update Kurs Emas Update kurs emas bisa anda dapatkan setiap hari, pada pukul 09.30 atau anda bisa menghubungi langsung ke PT Antam, Tbk.
  2. Perhatikan Dua Faktor Penentu yaitu faktor harga emas dunia dan faktor kurs rupiah terhadap dolar. Oleh karena itu disarankan untuk selalu meng-update setiap saat, informasi dua faktor tersebut.
  3. Perhatikan Keaslian Emas Keaslian emas dapat mengacu pada sertifikat yang diperoleh pada saat transaksi emas batangan (lempengan). Dimana sertifikat itu harus dikeluarkan oleh PT Antam, Tbk. (khusus Indonesia) yang berstandar internasional dan telah diakui oleh London Bullion Market Association (LBMA). Sertifikat asli memiliki nomor seri yang juga terdapat pada lempengan emas, dan ukuran 5 x 6 cm. Sedangkan untuk memastikan keaslian emas lempengan, anda dapat melihat logo LM berbentuk segi lima yang tertera, serta terdapat tulisan Fine Gold .9999, dan apabila nilai emas lebih dari 5 gram disertai nomor seri pada lempengan (yang biasanya diawali dengan dua karakter huruf dan tiga digit angka).
  4. Pastikan Kadar Kemurnian Emas, sesuai dengan Standar Internasional Emas 24 Karat (emas murni) berkomposisi 99.99% emas, Emas 22 Karat berkomposisi 91.7% emas dan 8.3% campuran bahan lain (perak), Emas 20 Karat berkomposisi 83.3% emas, Emas 18 Karat berkomposisi 75.0% emas, Emas 16 Karat berkomposisi 66.6% emas, Emas 14 Karat berkomposisi 58.5% emas, dan Emas 9 Karat berkomposisi 37.5% emas.
  5. Biaya Produksi Biaya produksi yang dikenakan berkisar antara Rp 33.500,- sampai dengan Rp 102.000,-/keping emas.
  6. Simpan Bukti Pembelian dan Bukti Keaslian Emas Hal ini adalah sebagai bukti keaslian bilamana anda menjual emas kembali ke tempat Anda membelinya, karena tentu akan lebih mudah dan tidak ada banya pertanyaan seputar emasnya.




    Posted in: Emas Batangan, Investasi Emas, logam mulia.
    Tagged: Cek Keaslian Emas · emas 14 karat · emas 18 karat · Emas 22karat · emas 9 karat · Investasi Emas · investasi logam mulia · Kadar Kemurnian Emas · keping emas · Kurs Emas · logam mulia · London Bullion Market Association · PT Antam · tips investasi

Wednesday, April 20, 2011

Switching Careers - 7 Key Steps By Alotta Candor Article Source: EzineArticles.e



Are you thinking about switching careers? If you are, you're not alone. Most Americans switch careers three times in their lifetime. Nevertheless, switching careers is scary. And it's especially paralyzing the older you get. But making a career switch is very possible and much more common than you might think. Before you're ready to leap, realize that it's a heavyweight decision that deserves some time and solid thought. Here are seven steps to help you on your way.

1. Gain insight from your current situation.

When considering a career switch, the first thing you should do is learn from your current situation. To do this, take a step back and study what you do for a living today and why you do it. Examine the reasons that you are in your current job or career. Was it what you went to school for? Was it what your parents wanted you to do? Was it the "hot career" at one time? Did you just "fall into it"? Did you love it at one time? Did you do it for the money you could make? Was it just to pay the bills? The answers to these questions can provide valuable insight into the core reasons that you want or need to change.

Now examine why you want to leave your current career field. Remove any company or management related politics that are specific to your current employer from the picture. See your situation for what it is and ask yourself why you are looking to switch. Are you being forced out because of market shifts of business trends? Are you burnt out? Do you want to make more money? Are you miserable doing what you do? Have you tried your best but found that your career is "just not a good fit"? Have you decided its time to pursue a long lost career love?

Inspecting your current situation and reasons for your desire to change careers will provide a foundation for your next step.

2. Look inside

Whether you already have a career in mind or you are searching for a new career, you must look inward. In order to gain the most from your reflection, it is essential to start with a clean slate. Set aside any notions (real or imagined) about what type of money certain careers offer. Discard any stereotypes or judgments of occupations. Distance yourself from any pre-conceived ideas about what you are right or destined for.

Now seriously examine what you truly love. First start with the obvious. Look at your hobbies and interests. List out the things you are passionate about or in which you have talent. Give yourself credit for things you are good at and don't be afraid to write things down that you love, but are not yet good at. Write them all down, even if you think they may not be a possible career path. You're just brainstorming at this point and you should not eliminate anything right out of the gate. And it's important to bear in mind that what you may think are your interests are not necessarily all of your interests. To help you get a good look at your interests, observe the simple things. What kinds of news stories perk your interest? What kinds of TV shows do you enjoy? What kinds of books magazines do you find yourself drawn to? What kind of people do you like to associate with or find interesting? What parts of your current career have brought you the most satisfaction?

Biology Terms: Countershading By Gwen Nicodemus Diamond Quality Author Article Source:



Many marine creatures are countershaded, which is a type of camouflage for them. What exactly is countershading?

Imagine that you are in a boat on the ocean and looking down. The ocean water looks dark blue. If you were an animal in the ocean, what color should you be to hide from animals in the sky, or higher up in the ocean, than you are?

Now, imagine you are SCUBA diving on the bottom of the ocean floor and look up, toward the sky. During the daytime, the sunlight filters into the water. What color should an animal be so it can blend into the water and hide from the animals below it in the water?

Animals selected for countershading

Animals that are darkly colored on their top part and lightly colored on their underside are said to be countershaded.

Animals that are darkly colored on their top part, where their dorsal fins are located, are less likely to be found and eaten by sky creatures or ocean animals higher up in the ocean than they are. Their dark colored skin blends into the color of the ocean around them and they have higher survival rates than animals that are lightly colored on the top.

Fun Science Experiment For Kids - How To Make Giant Bubbles! By Kalpana Nair


Fun Science Experiment For Kids - How To Make Giant Bubbles!
By Kalpana Nair



Fun Science experiments are not only liked by children but adults as well. Personally, I have tried many Science experiments when I was a kid and later on with my own family members. Making giant bubbles is an absolute favorite of mine and you can do this experiment too in a matter of minutes.

Things You Will Need

In order to make giant bubbles, you need to get your hands on the following items -

1. Washing liquid.
2. Glycerine or corn syrup.
3. Jug
4. Water.
5. A large bucket.
6. A spoon.
7. A wire coat hanger.
8. A ball of string.
9. An electrical tape.

How To Make Your Own Giant Bubbles

The Biotechnology Industry By Richard Graydon, M.D.



The primary goal of the biotechnology industry is to invent new biologically-active substances, for the treatment of disease, the pharmaceutical industry, and the management of agriculture.

The U.S. is the world leader in biotechnology, currently providing employment for more than 300,000 people in over 6,000 U.S. biotech companies, with an estimated market capitalization of approximately USD 281 billion in 2008. The biotechnology industry has more than tripled in size since 2000, with revenues increasing from USD 25 billion in 2000 to more than USD 80 billion in 2008. In 2007, Amgen, Inc., the world's largest biotechnology company, achieved total sales of USD 14.7 billion. Expectations are that the biotechnology market will increase to more than USD 130 billion by 2011.

Geographically, the U.S. accounts for 65% of the biotechnology market, Europe 23%, Canada 7%, Australia 3%, and the rest of the world, 2%.

The 10th Annual Multi-Unit Franchising Conference.

The 10th Annual Multi-Unit Franchising Conference.

For ten years, the Multi-Unit Franchising Conference has been the premier conference for multi-unit franchisees. This annual event continues to be the nexus point of learning, networking, and deal-making for multi-unit franchising.
THIS YEAR'S CONFERENCE - WHAT'S NEXT

We all know how to improve our performance by learning from the past. But today's complex business decisions require the ability to see what's on the horizon and anticipate what's next. At this year's conference we'll explore the changes and trends affecting the future of multi-unit franchising and the strategies you need to set in motion today to maintain your competitive advantage.
KEYNOTE SPEAKERS


Saturday, April 16, 2011

Special Seminar: Agricultural Biotechnology in Indonesia

Thu, January 13, 2011. 12:00-13.30
Prof. Hadi Susilo Arifin attended a Special Seminar of Agricultural Biotechnology in Indonesia
By Dr. M. Herman, Senior Scientist, ICABIOGRAD Biotechnology Center, Bogor, Indonesia.
Agenda
Day/Date: Thursday, January 13, 2011
Time: 12:00 noon – 1:30 p.m.
Venue: 271 Plant and Soil Sciences Building, Michigan State University
Seminar is organized by: WorldTAP/College of Agriculture and Natural Resources and ISP/Asian Studies Center
Pizza Lunch will be served.
Prof. Hadi Susilo Arifin and Dr. Muhammad Herman are colleagues in the team member of POKJA Ahli – Dewan Ketahanan Pangan Nasional RI (Taskforce Expertist - National Board of Food Security, the Republic of Indonesia) – 2010/2013.  In the end of the seminar, they have talked together regarding collaboration program in education and capacity building among MSU – IPB – UNPAD. Through an informal discussion, Prof. Karim (Biotechnology – MSU), Prof. Siddharth Chandra (Director of Asian Studies Center), and Prof. Hadi Susilo Arifin (IPB) have expected that for the future collaboration in education and capacity building between MSU and IPB should be expanded not only in biotechnology, but included climate change, environmental management and other actual issues. Some modules and curricula could be established together and delivering them for short courses, workshops, or summer school. These programs should be involved more universities in Indonesia.

Friday, April 1, 2011

How to Choose Speaker

This article can be used for reference in selecting speakers. Choosing speakers is tricky. Easy in the sense that the speaker now has a quality that is reliable. difficult, too many outstanding speakers, So making our  confused to choice. Therefore here I will share some experience about choosing the speakers.
Here are some points to  selecting the speakers:

How to Choose Speaker

  1. Set option for what the speaker to used, whether used indoors or outdoors, because of different types. For speakers who used outside the room, select the type of full range of means all tone is created by the speakers. By using this type of full range, a tone that is created will not be lost, either low or high-pitched tone. But the shortcomings, the speakers full-range tone that created less tender. Meanwhile, if you want to buy speakers for use in the room chose the type woofer (not a sub woofer, because different species), with the woofer low tone will be heard by more soft and smooth. If speakers are used outside the room then the low tone will be lost.
  2. The larger diameter of the speaker, the lower the tone that can be produced, but certainly require a large power amplifiers. 80-10 inches is suitable for home, but the size of 14 inches is not big enough to use a small stage. At least 18 inches, if you want to use in the room, then 10 inches is more than enough.
  3. The speakers that have a large magnet usually have good quality, but the price usually also follow, that meaning is more expensive.
  4. Look for speakers with great power. Do not be fooled by the big power contained labeled (sticker). Look more closely. There are speakers that include the ability to direct the magnetic power, but many were written on a paper sticker. Find a written directly in the magnet, because it is the original power of the speaker. You better choose the power of 150 watts is written directly in the magnet from the power of 300 watts but written on a paper sticker.
  5. The speakers will not produce good sound if it stands alone, so even if you buy any expensive speakers, if you will not be accompanied by Midle range and tweeter will be useless.
  6. large spool having a better quality and will produce a louder sound.
  7. Note the flexibility of the leaves of the speakers, the more flexible will usually produce a low tone more soft and smooth.
  8. Notice the brand, Look for brands that are trusted for speaker manufacturers.
So what can I share with you all, may be useful for visitors, thank you.

How to Choose Speaker

Wednesday, March 30, 2011

UNITED THERAPEUTICS IS AMONG THE COMPANIES IN THE BIOTECHNOLOGY INDUSTRY WITH THE HIGHEST EPS GROWTH (UTHR, GENZ, ALXN, CELG, CEPH)

Mar 30, 2011 (SmarTrend(R) News Watch via COMTEX) -- Below are the top 5 companies in the Biotechnology industry ranked by the year-over-year expected EPS growth rate. The long-term growth rate is the expected annual increase in operating EPS over the next three to five years.
United Therapeutics (NASDAQ:UTHR) EPS is expected to grow 283.1% year-over-year, better than the company's long-term growth rate of 60%. Based on the forward P/E of 12.7x its PEG ratio is 0.21, which signifies a discount in value relative to growth.
Genzyme (NASDAQ:GENZ) EPS is expected to grow 234.8% year-over-year, better than the company's long-term growth rate of 19.3%. Based on the forward P/E of 18.6x its PEG ratio is 0.96, which signifies a discount in value relative to growth.
Alexion Pharmaceuticals (NASDAQ:ALXN) EPS is expected to grow 118.3% year-over-year, better than the company's long-term growth rate of 38.8%. Based on the forward P/E of 43.9x its PEG ratio is 1.13, which signifies a premium valuation given for growth.
Celgene (NASDAQ:CELG) EPS is expected to grow 60% year-over-year, better than the company's long-term growth rate of 25.7%. Based on the forward P/E of 16.4x its PEG ratio is 0.64, which signifies a discount in value relative to growth.
Cephalon (NASDAQ:CEPH) EPS is expected to grow 49.3% year-over-year, better than the company's long-term growth rate of 10.2%. Based on the forward P/E of 7x its PEG ratio is 0.68, which signifies a discount in value relative to growth.
SmarTrend currently has shares of Genzyme in an Uptrend and issued the Uptrend alert on July 23, 2010 at $60.20. The stock has risen 26.3% since the Uptrend alert was issued.

Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo

Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo

Chen Zhang1, Kun-zheng Wang1, Hui Qiang1, Yi-lun Tang1, Qian Li2, Miao Li2 and Xiao-qian Dang1
  1. 1Department of Orthopedic Surgery, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China
  2. 2Department of Ultrasound, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China
Correspondence: Xiao-qian Dang, E-mail dang_xiaoqian@sohu.com
Received 22 February 2010; Accepted 6 May 2010; Published online 28 June 2010.
Top

Abstract

Aim:

 
To investigate the therapeutic potential of adeno-associated virus (AAV)-mediated expression of vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP).

Methods:

 
Four experimental groups were administered the following AAV vector constructs: rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7 (AAV-VEGF/BMP), rAAV-hVEGF165-GFP (AAV-VEGF), rAAV-hBMP-7-GFP (AAV-BMP), and rAAV-IRES-GFP (AAV-GFP). VEGF165 and BMP-7 gene expression was detected using RT-PCR. The VEGF165 and BMP-7 protein expression was determined by Western blotting and ELISA. The rabbit ischemic hind limb model was adopted and rAAV was administered intramuscularly into the ischemic limb.

Results:

 
Rabbit bone marrow-derived mesenchymal stem cells (BMSCs) were cultured and infected with the four viral vectors. The expression of GFP increased from the 7th day of infection and could be detected on the 28th day post-infection. In the AAV-VEGF/BMP group, the levels of VEGF165 and BMP-7 increased with prolonged infection time. The VEGF165 and BMP-7 secreted from BMSCs in the AAV-VEGF/BMP group enhanced HUVEC tube formation and resulted in a stronger osteogenic ability, respectively. In rabbit ischemic hind limb model, GFP expression increased from the 4th week and could be detected at 8 weeks post-injection. The rAAV vector had superior gene expressing activity. Eight weeks after gene transfer, the mean blood flow was significantly higher in the AAV-VEGF/BMP group. Orthotopic ossification was radiographically evident, and capillary growth and calcium deposits were obvious in this group.

Conclusion:

 
AAV-mediated VEGF and BMP gene transfer stimulates angiogenesis and bone regeneration and may be a new therapeutic technique for the treatment of avascular necrosis of the femoral head (ANFH).

Keywords:

adeno-associated virus; vascular endothelial growth factor; bone morphogenetic protein (BMP); avascular necrosis of the femoral head (ANFH); gene therapy
Top

Introduction

Recent insight into the pathogenesis of avascular necrosis of the femoral head (ANFH) has not identified satisfactory methods to increase blood circulation in necrotic areas of the femoral head, to promote bone regeneration, or to prevent osteonecrosis. The rapid development of gene therapy technology is increasingly recognized as a new therapeutic option for the treatment of ANFH, especially through therapeutic neovascularization and bone formation. Among growth factors, vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP) play important roles and have been extensively studied.
The VEGF family of growth factors is one of the most important cytokine families involved in angiogenesis. These factors promote the division of vascular endothelial cells and induce angiopoiesis. VEGF growth factors are essential for bone formation and repair during the bone regeneration process, which directly attracts endothelial cells and osteoclasts and enhances the differentiation of osteoblasts1, 2. BMP growth factors are the only signaling molecules that are individually sufficient for the induction of bone formation at orthotopic and heterotopic sites. They have defined roles in stimulating the proliferation and differentiation of mesenchymal and osteoprogenitor cells and have efficient bone induction activity3, 4. Because bone formation is a coordinated process involving the BMP and VEGF growth factors5, 6, orchestrating the timing with which these two factors are expressed may greatly enhance this process.
Choosing a safe and effective vector system to transfer and correctly express a target gene during gene therapy is important. Several different strategies have been examined for the delivery of genes of interest, including the use of naked DNA or an adenoviral vector. Treatment with naked DNA is simple and well tolerated by the recipient organism due to its low toxicity and weak induction of immune responses. However, the transduction efficiency is significantly lower when compared with other methods. The adenovirus has frequently been the vector of choice for gene transfer because it is able to transduce a variety of cells with high efciency. However, adenoviral vectors have major limitations, including a lack of sustained expression, the antigenicity of viral proteins that are targeted by both humoral immunity and cytotoxic T lymphocytes, and possible toxicity at high doses. However, there are many inherent features of the adeno-associated virus system that make it an attractive option as a human viral vector. AAV is a non-pathogenic, defective human parvovirus that requires the presence of a helper virus, such as adenovirus or herpes virus, for productive infection7, 8. Other advantages of this vector system include its low immunogenicity, its ability to transduce both dividing and non-dividing cells, the potential to integrate into specific sites, its ability to achieve long-term gene expression (even in vivo), and its broad tropism, allowing for the efficient transduction of diverse organs9. These features make AAV attractive and efficient for gene transfer in vitro and local injection in vivo.
To enhance neovascularization and bone regeneration during osteonecrosis therapy, we constructed adeno-associated viruses co-expressing hVEGF165 and hBMP-7 (rAAV-VEGF165-IRES-BMP-7) and detected their effect on gene expression and biological activity in vitro and in vivo. These data demonstrate the synergistic action of these two genes and may provide a new therapeutic option for ANFH.
Top

Materials and methods

Materials and reagents

The rAAV-hVEGF165-IRES-hBMP-7 (AAV-VEGF/BMP), rAAV-hVEGF165-GFP (AAV-VEGF), rAAV-hBMP-7-GFP (AAV-BMP), and rAAV-IRES-GFP (AAV-GFP) plasmids were constructed by Dr Xiang-hui HUANG. Human embryonic kidney cells-293 (HEK-293) and human umbilical vein endothelial cells (HUVECs) were obtained from the Department of Orthopedic Surgery in the Second Affiliated Hospital of Xi'an Jiaotong University. Male New Zealand rabbits (two months old, weighing 2.0–3.0 kg) were obtained from the experimental animal center of Xi'an Jiao Tong University. All animal protocols followed the recommendations and guidelines of the National Institutes of Health and were approved by the Xi'an Jiao Tong University Animal Care and Use Committee. The AAV helper-free system was obtained from Stratagene (La Jolla, CA, USA). A schematic representation of the structure of the plasmids in the AAV Helper Free System is provided in Figure 1A

Sunday, March 27, 2011

Insanity Workout - Pushing Workout To The Extreme

Are you sick and tired of the standard workout and diet that does not cause you to lose weight whatsoever? Then how about using a different sort of exercise method that will definitely make you shed the unwanted fats you long to take away for years. Shaun T’s insanity workout could be the answer to your desired body figure. It’s a 60-day workout program that will absolutely help you get the body weight and figure you’ve been wishing for in years. Compared to the other intense routines out in the market, this program only takes shorter time however still enable you to do the extensive movements that will ensure to make you sweat off the extra fat away. This program will be suitable for those who rarely have the time and chance to visit the gym.
insanity workout In case you are unhappy with shorter time frame of workout, you might try out something with similar intense and complex exercise moves which you could find in px90. It’s a 90-day intense workout program created by Tony Horton. The said program includes intense workout, complex actions that needs the use of resistance bands and yoga mat to further attain your weight goal. Both this programs will surely help you obtain the body figure and weight you always wished to have. insanity workout,

Insanity Workout - Pushing Workout To The Extreme

Saturday, March 19, 2011

Cell therapy strategies and improvements for muscular dystrophy



Edited by R De Maria

M Quattrocelli1, M Cassano1, S Crippa1, I Perini1 and M Sampaolesi1,2

1. 1Translational Cardiomyology, SCIL Katholieke Universiteit Leuven, Herestraat 49 bus 814, Leuven 3000, Belgium
2. 2Human Anatomy, University of Pavia, Via Forlanini 8, Pavia 27100, Italy

Correspondence: M Sampaolesi, Translational Cardiomyology, Stem Cell Research Institute, University Hospital Gasthuisberg, Herestraat 49, Leuven B-3000, Belgium. Tel: +32 0163 30295; Fax: +32 0163 30294; E-mail: maurilio.sampaolesi@med.kuleuven.be

Received 9 July 2009; Revised 8 September 2009; Accepted 21 September 2009; Published online 30 October 2009.
Top of page
Abstract

Understanding stem cell commitment and differentiation is a critical step towards clinical translation of cell therapies. In past few years, several cell types have been characterized and transplanted in animal models for different diseased tissues, eligible for a cell-mediated regeneration. Skeletal muscle damage is a challenge for cell- and gene-based therapeutical approaches, given the unique architecture of the tissue and the clinical relevance of acute damages or dystrophies. In this review, we will consider the regenerative potential of embryonic and somatic stem cells and the outcomes achieved on their transplantation into animal models for muscular dystrophy or acute muscle impairment.
Keywords:

muscular dystrophy; animal models; cell therapy; stem cells
Abbreviations:

ABCG2, ATP-binding cassette, sub-family G (WHITE), member 2; ALP, alkaline phosphatase; αsg, α-sarcoglycan; bg, natural killer (NK) cell-deficient mice (beige); BM, bone marrow; BMD, Becker muscular dystrophy; BMSCs, bone marrow-derived stem cells; BMT, bone marrow transplantation; βsg, β-sarcoglycan; c-MYC, cellular myelocytomatosis oncogene; cDNA, complementary DNA; Cxcr4, chemokine (C-X-C motif) receptor 4; DMD, Duchenne muscular dystrophy; ESCs, embryonic stem cells; FACS, fluorescence-activated cell sorting; Flk1, fetal liver kinase 1; GFP, green fluorescent protein; GLP–GMP, good laboratory practice–good manufacturing practice; GRMD, Golden Retriever muscular dystrophy; Gy, Gray (unit); HCT, haemopoietic cell transplantation; hMADS, human multi-potent adipose-derived stem cells; HLA, human leukocyte antigen; Hmgb1, high mobility group box 1; IGF1, Insulin-like growth factor 1; iPS, induced pluripotent stem cells; KLF4, Kruppel-like factor 4; KO, knockout; KSN, mice strain with high natural killer activity; LGMD, limb-girdle muscular dystrophy; MABs, mesoangioblasts; Mac1, integrin alpha M; MagicF1, cMet-activating genetically improved factor1; MAPCs, multipotent adult progenitor cells; mdx, muscular dystrophy X-linked (?); μDYS, micro-dystrophin; MGF, mechano growth factor; MMP9, matrix metallopeptidase 9; mRNA, messenger ribonucleic acid; MyoD, myoblast determination protein; Myf5, myogenic factor 5; NCAM, neural cell adhesion molecule; NG2, chondroitin sulfate proteoglycan 4; NICD, Notch1 intracellular domain; NOD, non-obese diabetic; OCT4, octamer-binding transcription factor 4; Pax3, paired box gene 3; PDGFRα, platelet derived growth factor receptor alpha; PIGF, phosphatidylinositol glycan anchor biosynthesis, class F; PTC124, 3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid (C15H9FN2O3); RT-PCR, retrotranscription-based polymerase chain reaction; Sca1, stem cell antigen 1; scid, severe combined immuno-deficiency; SDF1, stromal-derived factor 1; SMPs, skeletal muscle precursors; SOX2, SRY (sex determining region Y)-box 2; SP, side population; TNF-α, tumor necrosis factor-α; VEGFR2, vascular endothelial growth factor receptor 2; Wnt3a, wingless-type MMTV integration site family, member 3A

Muscular dystrophies are a heterogeneous group of inherited diseases, primarily characterized by severe and chronic skeletal muscle degeneration. Duchenne muscular dystrophy (DMD) is the most severe disease among similar dystrophic diseases and is caused by frame-shift deletions, duplications, or point mutations in dystrophin gene. Patient mobility is highly affected, usually resulting in wheelchair dependency, and death occurs due to respiratory or cardiac failure.1, 2

New strategies for the treatment of this disease are currently being investigated and are categorized by two approaches: endogenous activation and exogenous delivery (Figure 1). The first strategy consists of re-activating endogenous cells to achieve muscle hypertrophy, counteracting the mass/force loss in inflamed fibres. To reach this goal, a growing range of small molecules or recombinant proteins has been tested, including insulin-like growth factor 1 (IGF1),3, 4 MagicF15 or valproic acid.6 The second strategy, on the contrary, relies on exogenous tools (gene and/or cell therapies) to improve muscle regeneration, thus providing new, functional fibres to the dystrophic muscle. Gene therapy targets the genetic defects, attempting to overcome pathological mutations by providing the muscle with the correct form of the gene7 or by correcting the splicing through the exon-skipping vectors8, 9 or drugs, such as PTC124.10 Cell therapy, however, is based on stem cell-driven muscle regeneration, by systemic or local injections.
Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Combining endogenous activation and exogenous delivery to enhance muscle regeneration. Currently, two therapeutical approaches are investigated for the regeneration of the skeletal muscle: the endogeneous activation of physiological repair potential, such as targeting of satellite, circulating or perycite cells through small molecules, or the exogenous delivery of stem cells or genetic tools, such as antisense oligonucleotides (ODNs), drugs (PTC124) or viral vectors. M-SP, muscle side population; BM-SP, bone marrow side population; BMSCs, bone marrow-derived stem cells; MABs: mesoangioblasts; MADS, multipotent adipose-derived stem cells; iPS, induced pluripotent stem cells, not yet injected in animal models for muscular dystrophy; ESCs, embryonic stem cells
Full figure and legend (192K)

This review will focus on in vivo cell therapy strategies and improvements in the treatment of sarcoglycan/dystrophin complex-related dystrophies, such as Duchenne or limb-girdle muscular dystrophies.
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Animal Models for Sarcoglycan/Dystrophin Complex-Related Muscular Dystrophies

Sarcoglycan/dystrophin complex-related muscular dystrophies are caused by disruption of the sarcoglycan–dystrophin complex that normally anchors the actin fibres to the sarcolemma, generally resulting in chronic muscle wastage, progressive fibrotic infiltrations and force decrease. Moreover, cardiac involvement is often described, in terms of chronic dilatative cardiomyopathy, scar infiltrations, aneurisms and repeated microinfarctions.

DMD is the most severe form and it is caused by frame-shift mutations or huge deletions in the dystrophin gene. It is one of the largest gene in the human genome and encodes a 14-kb mRNA. In this type of dystrophy, the protein is completely or partially lost. A less severe phenotype is observed in Becker dystrophy, in which mutations still affect the dystrophin gene, but myofibres retain a truncated and low-active isoform of the protein. Some forms of limb-girdle muscular dystrophy (LGMD2) are also caused by mutations in sarcoglycan complex proteins, for example, α- or β-sarcoglycan (αsg or βsg) depletion, and can result in severe pathological phenotypes.

Several animal models have been developed to study muscular dystrophies, particularly for DMD and LGMD2. The most widely used model for dystrophy is the mdx mouse that carries an X-linked mutation in the dystrophin gene, thus mimicking, at least in principle, the DMD genotype in humans. In mdx mice, the effects of degeneration are less severe, mainly due to the presence of relatively high numbers of revertant fibres (1–3%)11 and an upregulation of utrophin. Utrophin is a smaller analogue of the dystrophin and may account for the partial compensatory effect on muscle wastage.12, 13

Recently, it has been demonstrated that mdx satellite cells undergo telomere erosion, which may also contribute to the inability of these cells to continuously repair DMD muscle. It is possible that muscle stem cells or myogenic progenitors that maintain telomerase activity until late passages, may contribute in part to the muscular regeneration, that provides mdx mice with a normal life span.14

Feasible models of LGMD2 are α-sarcoglycan- and β-sarcoglycan-knockout mice. These mice are very close to the human phenotype of LGMD2D and LGMD2E, respectively, as they show chronic skeletal muscle degeneration and, in the case of βsg-KO mice, dilatative cardiomyopathy.15, 16 In α-sarcoglycan-KO mice, αsg gene is disrupted through a neomycin cassette insertion between exons 1 and 9, through homologous recombination of the flanking regions. Similarly, in the β-sarcoglycan-KO mice, the region encompassing exons 3–6 of βsg gene is disrupted. LGMD mice are considered a better animal model than mdx mice because of their lack of revertant fibres, which often render mdx mice-related results controversial.17

Canine models of DMD are being also extensively studied.18, 19 The larger fibres in canine muscles mimic the human dystrophy effects better than that of the mouse. At present, there are two major colonies of dystrophic dogs all over the world, bearing the same mutation in different genetic backgrounds; a colony of Golden Retrievers and one of Beagle dogs. These animals are both derived through cross-breeding of a naturally born, affected founder because transgenic creation would be unethical in these animal models. The mutation lies in intron 6 of dystrophin gene and results in aberrant splicing that causes a premature transcription stop codon. Dystrophic dogs, from both varieties, show extremely affected motility, posing, salivation, severe chronic scar infiltrations and skeletal muscle degeneration. In these animals, revertant fibres are also almost undetectable, thus providing a good model to analyze regeneration effects of cell and genetic therapies.
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Cell Models for In Vivo Skeletal Muscle Regeneration

To date, the only clinical treatments for muscular dystrophy are steroid administration and non-invasive intermittent positive pressure ventilation. These treatments result in amelioration of symptoms and improved quality of life.20 Despite the benefits of slightly longer lifespan, alleviation of pain and surgical management of scoliosis, this therapy shows many side effects, such as weight increase, and has no real beneficial effects on skeletal muscle architecture and force.21 Thus, cell therapy represents a theoretical valuable alternative. The main goal of cell therapy is to directly regenerate wasted, adult muscle fibres through systemic or targeted injection of stem cells, which function to block muscle loss and restore, at least partially, the normal muscular activity. It is currently a difficult task to conjugate high efficiencies in cell motility, homing, engraftment and differentiation into the complex environment of a severely inflammed and degenerated muscle. Several cell models have been tested in vivo, with diverse results (Table 1). Three main classes of stem cells with a myogenic differentiation potential have been considered for cell therapy protocols in preclinical studies for the treatment of muscular dystrophy: (i) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSs); (ii) bone marrow-derived stem cells (BMSCs) and circulating progenitors; and (iii) local myogenic-committed progenitors.
Table 1 - Stem cell types for the treatment of chronic or acute skeletal muscle damage.
Table 1 - Stem cell types for the treatment of chronic or acute skeletal muscle damage - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorFull table

ESCs and iPS cells

Embryonic stem cells (ESCs) are generally considered the most promising natural source of pluripotent cells for cell therapy, but few attempts have been reported in muscle regeneration and efficiencies are still quite low. Importantly, ethical issues around their procurement and use, such as blastocyst disgregation and oocyte requirement, have raised a lot of concerns and limited the use of human ESCs (hESCs) for research purposes in several countries.

The critical step in using ESCs resides in cell conditioning before engraftment, a necessary step that increases the differentiation rate towards myogenesis and avoids teratoma formation. A heterogeneous suspension, derived from a co-culture of male embryoid bodies and female freshly isolated dystrophic satellite cells, has been demonstrated to produce few chimeric, dystrophin-positive myofibres in injured muscles of female mdx mice.22 Promising results have been obtained with hESCs, cultivated to enrich in mesenchymal precursors. The CD73+ NCAM+ sub-fraction was able to differentiate into myotubes in vitro and, remarkably, to regenerate up to 7% of injured skeletal muscle in immunodeficient mice.23 Recently, another strategy has been developed using paraxial mesoderm progenitors that are isolated from differentiating ESCs. The PDGFRα+/Flk1− fraction of Pax3-induced, embryoid body-derived cells shows activation of myogenic transcription factors in vitro and good differentiation in dystrophin+ fibres on transplantation into both cardiotoxin-injured and dystrophic muscle. Injected mice also show an amelioration of the contractility force. In this case, intra-arterial administration of cells resulted in higher engraftment than intravenous injection.24 In another study, paraxial mesoderm progenitors were isolated as PDGFRα+/VEGFR2+ cells, which also have been successfully tested in cardiotoxin-injured quadriceps of KSN nude mice.25

A new source of pluripotent cells comes from the reprogramming of adult murine or human somatic cells, by means of pluripotency transcription factor expression. Human iPS (induced pluripotent stem, hiPS) cells are reprogrammed from differentiated, adult cells, such as fibroblasts, to an ES-like status, by retroviral-mediated transduction of OCT4, SOX2, KLF4 and, even if dispensable, c-MYC.26, 27, 28 As hiPS cells are created by reprogramming adult cells into a flexible, embryonic-like state, they have been claimed as alternative pluripotent cells to overcome the ethical issues regarding the use of ESCs. However, some reservations exist regarding the in vivo safety of hiPS cells, which must be addressed in the future. Recently, a wide range of disease-specific hiPS cells has been generated from patients with various Mendelian or complex diseases, including Duchenne and Becker muscular dystrophy.29 These cells could represent a greater advancement in terms of plasticity and life span, in comparison with other cell lines tested to date. Moreover, these DMD- or BMD-specific hiPS cells show the same genetic background of the donor, thus representing a good model for in vitro drug testing and, if genetically corrected, a suitable cell line for extensive skeletal muscle repair.
Bone marrow-derived and circulating progenitors

Mesenchymal stem cells have been tested in acute and chronic muscle wastage, but results are still controversial. After bone marrow transplantation (BMT) in dystrophic mice, BMSCs are able to migrate and contribute to the formation of new Myf5+ fibres through repeated rounds of inflammation and regeneration, which is typical of the mdx mouse muscles.30 In humans, the clinical case of a young DMD patient (deletion of the exon 45 in the dystrophin gene) has been reported, which 12 years after BMT, showed donor nuclei fused to 0.5% of dystrophic myofibres.31 Real-time RT-PCR analysis detected a small amount of a truncated, in-frame isoform of dystrophin, lacking exons 44 and 45, and trace amounts of the wild-type gene (0.0005%), although a direct correlation between the BMT-derived nuclei and the dystrophin isoform expression was missing. In addition, human mesenchymal stem cells isolated from synovial membrane of adult donors, on intramuscular delivery into tibialis anterior of mdx mice, efficiently produce new, functional myofibres, without any sign of fusion. These cells also contribute to the long-term satellite cell population and restore mechano growth factor (MGF) expression in treated muscles.32 The myogenic potential of these cells seems to be strongly related to the microenvironment surrounding the delivered cells because when injected systemically in dystrophic mice, they are observed in almost all tissues.

Given the broad availability of the source, human multipotent adipose-derived stem cells (hMADS) have been investigated as a possible alternative for muscle regeneration. hMADS are CD44+, CD90+ and CD105+, confirming their mesenchymal lineage. Once injected intramuscularly into tibialis anterior of immunocompetent and immunosuppressed mdx mice, they fuse with host fibres, resulting, 6 months later, in a large number of chimaeric myofibres expressing human dystrophin. Interestingly, no differences are reported between immuno-competent and immuno-suppressed hosts and it also seems that hMADS significantly reduce necrosis in the dystrophic muscle.33 These promising results have since been enhanced by priming the adipose-derived mesenchymal cells by co-culturing with myoblasts34 or forced MyoD expression.35

A sub-population of circulating, haematopoietic stem cells expressing CD133, which constitutes another interesting and easily isolatable cell pool, has been reported to express early myogenic markers.36 Intramuscular or intra-arterial injection of genetically corrected CD133+ progenitors, isolated from both peripheral blood and muscles of DMD patients, results in a significant recovery of muscle morphology, function and re-expression of human dystrophin in scid/mdx mice.37

In contrast, several studies have reported of absent or incomplete muscle repair by mesenchymal or haematopoietic stem cells. On being intravenously injected into a mouse model for LGMD2F (mice lacking δ-sarcoglycan), bone marrow side population cells engraft and fuse into skeletal fibres, but do not restore δ-sarcoglycan expression.38 Green fluorescent protein-positive bone marrow (GFP+ BM) cells, delivered through retro-orbital injection, fuse with ~3% of fibres in the tibialis anterior of treated mdx mice, but almost no dystrophin expression is detected in GFP+ myofibres. However, where dystrophin is detected, its expression is spatially more limited than in revertant fibres.39 These data have been confirmed in another study, in which it has been demonstrated that greater than or equal to80% of BM-derived muscle-incorporated nuclei in the transplanted dystrophic mouse are ‘silent’. Incorporated nuclei fail to express myogenic factors, including dystrophin, and this ‘silencing’ is still retained even in the presence of strong chromatin-remodelling agents, such as 5′-azacytidine.40 It has also been demonstrated that haematopoietic cell transplantation (HCT) alone result neither in any skeletal fibre regeneration nor in expression of dystrophin or other muscle genes.41 Nevertheless, an interesting role for HCT in muscle regeneration may come from immunotolerance effects towards allogeneic myoblast engraftment. DMD dogs have been successfully treated using both peripheral HCT and freshly isolated myoblasts from the same healthy donor. Donor myoblast-related dystrophin expression increased up to ~7% of wild-type levels and was maintained for at least 24 weeks, without any pharmacological immunosuppression.42

Finally, bone marrow-derived multipotent adult progenitor cells (MAPCs) were observed to durably repair muscles in ischaemic limbs, by efficient revascularization of necrotic tissues.43
Local myogenic-committed progenitors

Satellite cells are quiescent unipotent myoprecursors, located between the fibre and the basal lamina; during embryogenesis, they form during the second wave of myogenesis and, after contributing massively to the first post-natal muscle growth, they stop proliferating and reach their niche.44 They can also be re-activated on muscle damage, re-entering cell cycle and contributing to the formation of new muscle fibres.45 Given their natural commitment, it's easy to imagine satellite cells as a major candidate for muscle regeneration in muscular dystrophies. On single fibre transplantation into radiation-ablated mdx tibialis anterior, donor satellite cells multiply and expand, re-populating the satellite cell pool and differentiate into functional myofibres.46 Pax7+ CD34+ GFP+ satellite cells, isolated from Pax3GFP/+ mice diaphragms, have also been demonstrated to be a good cell model for mdx irradiated muscle treatment, resulting in the restoration of dystrophin expression in many skeletal fibres and contributing to the resident satellite compartment.47 Injections were administered intramuscularly and, notably, two major problems arose; satellite cells have a very low migration capability and, furthermore, cells showed impaired engraftment capability when expanded in vitro, even if for few days.47

To test the regeneration capability of satellite cells at a clonal level, single-cell dilutions of CD34+ integrinα+ luciferase-expressing satellite cells were injected into the skeletal muscle of a NOD/SCID mouse depleted of resident satellite cells by 18 Gy irradiation. It was shown by in vivo imaging that a single satellite cell can reconstitute the satellite compartment and, on further damage, can rapidly re-enter a new proliferation wave, generating new myofibres.48

Recently, interesting findings resulted from a prospective isolation of skeletal muscle precursors (SMPs), consisting of a CD45− Sca1− Mac1− Cxcr4+ β1-integrin+ subset within the endogenous satellite compartment. When injected into cardiotoxin-injured muscles in immunodeficient mdx mice, SMPs robustly contributed to muscle regeneration (up to 94%) by fusing with pre-existing fibres or stimulating de novo myogenesis. Muscle histology and contractile force in treated mice were significantly better than those in untreated mice. Furthermore, SMPs contributed greatly to the endogenous satellite population, undergoing new cycles of re-activation on subsequent induced damage.49 However, as freshly isolated SMPs were injected, without in vitro expansion, the migration capability remains restricted in the areas surrounding the intramuscular injection site.

One of the major concerns about satellite cells is that, probably, they are not a homogeneous population. As argued by Collins et al.,46 the variable engraftment rate of satellite cells from a single myofibre transplantation could be due to the functional heterogeneity of the satellite cell pool and of their niche of origin. Satellite cell heterogeneity is still a contentious issue and has been extensively reviewed in several studies50, 51 and is still open for debate.52

Muscle side population (SP) cells are defined as Sca1+ CD45+ cells, able to rapidly efflux the Hoechst dye 33342, and are being investigated as potential myogenic progenitors. They are associated with the muscle vasculature and are spontaneously committed towards the haematopoietic lineage. On co-culture with myoblasts, they can form myotubes in vitro and, if injected intramuscularly into crushed tibialis of a scid/bg immunodeficient mouse, can give rise to up to 1% of regenerating fibres.53 The efficiency of SP-mediated muscle regeneration has been increased to 5–8% by injection into the femoral artery of mdx5cv DMD mice, resulting in Pax7 and desmin expression by donor cells, after extravasation and recruitment to inflammation sites.54 Impressive results have been obtained with the identification of a rare subset (0.25%) of SP cells, characterized by both satellite- and SP-related markers, such as Sca1+/ABCG2+/Syndecan4+/Pax7+, and found in the typical satellite compartment, under the basal lamina. Once sorted from the mononuclear fraction of the hind limb, they can grow in association with single muscle fibres and can robustly undergo myogenic differentiation in vitro. On intramuscular injection in the presence of 1.2% BaCl2, these satellite-SP cells have been shown to efficiently compete with endogenous satellite cells in regenerating the wild-type muscle. Injected cells resulted in 30% fibre regeneration and, strikingly, in up to 75% reconstitution of the endogenous satellite cell pool. The newly generated satellite cells were able to undergo new rounds of proliferation and muscle repair on subsequent injuries. Furthermore, the same long-term effects have been proven through injections into dystrophic mdx4cv tibialis anterior muscles, producing up to 70% regenerating fibres.55 However, BaCl2-induced muscle damage is not a widely used and accepted regeneration model, and this must be taken into consideration when interpreting these findings.

Recently, a new type of vessel-associated muscle-derived stem cells has been investigated as a suitable potential model for chronic muscle therapy, namely mesoangioblasts (MABs). They can be isolated from the dorsal aorta of E9.5 embryo56 or from adult skeletal muscle of mice, dogs and humans.57, 58, 59 MABs are CD34+, Sca1+, PDGFRα+, PDFGRβ+, NG2+ and ALP+, thus supporting the idea that they are a sub-group of the pericytic population.60 They show high proliferation rates in vitro, without transformation potential, and display, in in vitro and embryonic chimaera systems, multipotent differentiation capability towards myogenic, osteogenic, chondrogenic and adipogenic lineages. On intra-arterial injection into inflammated muscles of αsg-KO mice or DMD Golden Retriever dogs, they are capable of consistently regenerating (up to 50%) the muscle architecture, sarcoglycan/dystrophin expression and electrophysiological properties of wasted dystrophic muscle.57, 58 Similarly, good results have been achieved through human MAB transplantation into scid–mdx immunodeficient dystrophic mice.59

Treatment of dogs with MABs resulted in very good and long-term results in some animals, in terms of general motility restoration and whole muscle regeneration, whereas other animals did not show a good engraftment and any clinical improvement.58 This confirms the general idea that background variability in dystrophies in larger organisms, such as dogs or humans, has to be evaluated for effective cell therapies.

Furthermore, another class of myogenic precursors has been isolated from endothelial population of adult human muscle, through FACS-mediated prospective isolation of CD56+ CD34+ CD144+ cells. These myoendothelial progenitors, on injection into injured muscle of scid mice can achieve muscle engraftment and fibre neo-formation at an higher degree than CD56+ canonical myoprecursors.61
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Cell Conditioning and Priming

To increase engraftment specificity and differentiation potential, cell transplantation can be combined with pre-injection cell conditioning or with genetic manipulation.

Cell migration limitations are often a major cause of low engraftment efficiencies in skeletal muscle, which is a very complex tissue with severely limited cell motility, particularly in the presence of large fibrotic or necrotic areas. Thus, some attempts have been made to assist migration by conditioning cells with migration-enhancing soluble factors or by overexpressing commiting/mobilizing proteins before applying them to the degenerated muscle.

A lot of interest has been shown in Notch signalling, as a potential enhancer of myogenic commitment. Rat BMSCs, transfected with a plasmid for the intracellular domain of Notch1 (NICD) and injected locally or intravenously into injured muscles of rats or nude mice, account for a very high level of regenerating fibres (up to 89%).62 Given that NICD is the active signalling form of the receptor, it could be possible that finer genetic tools to activate Notch signalling could enhance myogenesis by donor cell injection, although recently it has been demonstrated that a temporal switch from Notch to Wnt3a signalling activation is necessary during normal adult myogenesis.63

Encouraging in vivo results have come from cell therapy experiments involving soluble factor-dependent cell conditioning. Hmgb1, a cytokine secreted by activated macrophages and monocytes, is able to increase the recruitment of MABs out of the vessels into the muscle. Heparin–Sepharose beads, loaded with Hmgb1 and injected into the femoral artery, were shown to promote the trans-endothelial migration of intra-arterial-injected embryonic MABs into non-injured tibialis anterior of wild-type mice.64 Pre-treatment of MABs with mobilization cytokines, such as SDF1 and TNFα, highly increase MAB homing into dystrophic muscle of αsg-null mice, thus reducing approximately 50% the aspecific homing into filter organs. Their regenerative effect was magnified by TNFα priming and α4-integrin overexpression.65 Furthermore, improving the angiogenic potential in necrotic areas could help cell therapy. For example, tendon fibroblasts expressing placenta growth factor (PIGF, an angiogenic factor) and matrix metalloproteinase 9 (MMP9), injected intramuscularly into aged αsg-KO mice, result in a dramatic increase in the extension of regenerated dystrophin+ muscle areas after intra-arterial delivery of wild-type MABs.66 Such results corroborate the idea that a deeper knowledge of cytokines and angiogenic factors regulating the inflammation-dependent recruitment of myoprecursors will serve to improve the benefits mediated by cell therapy.
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Genetic Manipulation for Autologous Cell Therapy

Correction of the dystrophic genotype in the transplanted cells could allow the usage of autologous cells, instead of heterologous wild-type cells, thus avoiding immunosuppressive drugs.

A widely used strategy relies on lentiviral transduction of muscle-regenerating cells to allow integration and expression of the disrupted gene. To correct DMD, several alternatives of the dystrophin gene (which is too long to be introduced in a lentiviral vector) exist, such as micro- or mini-dystrophin. These are shorter isoforms of the native protein, which retain a partial functionality, thus providing, in principle, a possible molecular rescue on differentiation towards newly formed myofibres. mdx5cv mice, injected intravenously with autologous muscle-SP cells that were previously transduced with human micro-dystrophin (hμDYS)-expressing lentiviruses, show some skeletal fibres positive for the human version of the lacking protein.67 Similarly, very good results came from transplantation of hμDYS-transduced human pericytes into mdx–scid mice.59 In GRMD dog treatment, autologous MABs, transduced with human micro-dystrophin, induced a quite widespread expression of dystrophin and other proteins of the sarcoglycan complex in analyzed muscles and a partial recovery of the histological architecture. However, treated dogs had poorly restored general motility and force.58 These results may support the idea that, especially in higher organisms, mini- or micro-genes are not so feasible, or, at least, show highly variable and only short-term effects. Genetic correction can be addressed also for treatment of other genetic defects resulting in dystrophy, as when αsg-KO MABs, carrying a lentiviral αsg cDNA construct under a constitutive promoter, are injected into LGMD2D mice, there is extensive fibre regeneration, mobility and muscle force recovery.57
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Conclusions and Perspectives

Stem cell-based therapy is the most attractive approach for the treatment of DMD and other muscular dystrophies, and research in this direction has moved rapidly in past few years. Experiments in small and large animal models are paving the way for clinical experimentation, but it would be imprudent to predict a ‘cure’ from these first attempts. Nevertheless, several clinical trials have been started or planned, involving myoblast or perycyte injection from HLA-matched donors and, given the growing variety of possible myogenic progenitors in literature, the number will increase during the near future. Other clinical trials are focusing on gene- or antibody-based strategies, such as adeno-associated viruses carrying γ-sarcoglycan or μ-dystrophin and antibody-triggered myostatin blockade.21

Furthermore, encouraging results have come from clinical trials related to exon-skipping technology. Specific exons carrying mutations can be skipped by antisense oligonucleotides administration to restore the reading frame and result in the production of internally deleted, but functional dystrophin. Recently, two clinical trials involving two different drugs, AVI-4658 (developed by the MDEX Consortium, United Kingdom and manufactured by AVI BioPharma, Bothell, WA, USA) and PRO051 (developed by University of Leiden, The Nederlands in collaboration with Prosensa B.V.), were performed on Duchenne patients. In both trials, biopsy data showed that injections of antisense oligonucleotides, to skip exon 51, into dystrophic muscles, successfully induced new dystrophin production, with no adverse events. These pioneering studies are now followed by randomized controlled trials of systemic therapies both in The Netherlands and the United Kingdom.

Therefore, it is reasonable to expect encouraging results that may also drive a combination of the stem cell- and gene-based therapies. It is critical to better understand the biological properties of stem cells and their paracrine role in the treatment of muscular diseases, to realize the potential positive effects of these new cures.

The scientific community largely accepts the presence of adult stem cells in all tissues but their origin is still controversial. We and other authors suggest pericytes as a source of stem cells present in skeletal and cardiac muscles.59, 60 They are influenced by their surrounding when maintaining a specific cell commitment, although this has to be clarified in pathological tissues.

Interestingly, other research groups have identified mesenchymal cells as stem cells for all tissues,68 raising questions regarding the possibility that the primary source of cell plasticity is confined to the bone marrow. It could be possible that cells move from bone marrow towards pericyte compartment, to adopt a specific cell fate, influenced by local niche. To further elucidate their origin, it is necessary to generate transgenic animals to track endogenous stem cells during muscle development and regeneration.

Despite the fact that current regulatory restrictions will defer the clinical translation of new approaches, that are successful in animal models, several trials have been started. Moreover, the actual huge costs of GLP–GMP (good laboratory and manufacture practice) stem cell technology limit the feasibility of cell therapy treatment for patients affected by muscular dystrophy. However, strategies to lower costs are being investigated to develop treatments that are available for large numbers of patients. In conclusion, it is critical to better understand the biological properties of stem cells and their paracrine role in the treatment of muscular diseases, to realize the potential positive effects of these new cures. Therefore, it is reasonable to expect encouraging results from the on-going trials that may also drive a combination of stem cell- and gene-based therapies for the treatment of muscular dystrophies.
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References

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Acknowledgements

We thank Paolo Luban for his support. We are particularly grateful to Guido Tettamanti and Giulio Cossu for critical reading of the paper and for helpful comments, and Shea Carter for the ms proofreading service. This work was supported by FWO Odysseus Program n. G.0907.08; Wicka Funds n. zkb8720, University of Minnesota US; the Italian Ministry of University and Scientific Research (grant n. 2005067555_003, COFIN 2006–08), the Muscular Dystrophy Association, Association Francoise contre les Myopathies, CARIPLO Funds 2007-5639 and 2008-2005.

In vitro and in vivo multidrug resistance reversal activity by a Betti-base derivative of tylosin


N Gyémánt, H Engi, Z Schelz, I Szatmári, D Tóth, F Fülöp, J Molnár and P A M de Witte
Background:
The multidrug resistance (MDR) proteins are present in a majority of human tumours. Their activity is important to understand the chemotherapeutic failure. A search for MDR-reversing compounds was conducted among various Betti-base derivatives of tylosin.
Methods:
Here, we evaluate the in vitro and in vivo P-glycoprotein (P-gp)-modulating activity of the most promising compound N-tylosil-1-α-amino-(3-bromophenyl)-methyl-2-naphthol (TBN) using human MDR1 gene-transfected and parental L5178 mouse lymphoma cell lines.
Results:
In vitro experiments showed that TBN dramatically increased the P-gp-mediated cellular uptake of the fluorescent substrate rhodamine 123. Similarly, TBN was found to act as a very potent enhancer of the cytotoxicity of doxorubicin on the resistant cell line. We also provide in vivo evidence using DBA/2 mice in support for an increased tumoural accumulation of doxorubicin, without affecting its tissue distribution, resulting in an enhanced antitumoural effect.
Conclusion:
Our results suggest that TBN is a potent modulator of the P-gp membrane pump and that the compound could be of clinical relevance to improve the efficacy of chemotherapy in MDR cancers.

British Journal of Cancer 103, 178-185

Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo

Original Article

Acta Pharmacologica Sinica (2010) 31: 821–830; doi: 10.1038/aps.2010.67; published online 28 June 2010

Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo

Chen Zhang1, Kun-zheng Wang1, Hui Qiang1, Yi-lun Tang1, Qian Li2, Miao Li2 and Xiao-qian Dang1
  1. 1Department of Orthopedic Surgery, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China
  2. 2Department of Ultrasound, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China
Correspondence: Xiao-qian Dang, E-mail dang_xiaoqian@sohu.com
Received 22 February 2010; Accepted 6 May 2010; Published online 28 June 2010.
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Abstract

Aim:

 
To investigate the therapeutic potential of adeno-associated virus (AAV)-mediated expression of vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP).

Methods:

 
Four experimental groups were administered the following AAV vector constructs: rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7 (AAV-VEGF/BMP), rAAV-hVEGF165-GFP (AAV-VEGF), rAAV-hBMP-7-GFP (AAV-BMP), and rAAV-IRES-GFP (AAV-GFP). VEGF165 and BMP-7 gene expression was detected using RT-PCR. The VEGF165 and BMP-7 protein expression was determined by Western blotting and ELISA. The rabbit ischemic hind limb model was adopted and rAAV was administered intramuscularly into the ischemic limb.

Results:

 
Rabbit bone marrow-derived mesenchymal stem cells (BMSCs) were cultured and infected with the four viral vectors. The expression of GFP increased from the 7th day of infection and could be detected on the 28th day post-infection. In the AAV-VEGF/BMP group, the levels of VEGF165 and BMP-7 increased with prolonged infection time. The VEGF165 and BMP-7 secreted from BMSCs in the AAV-VEGF/BMP group enhanced HUVEC tube formation and resulted in a stronger osteogenic ability, respectively. In rabbit ischemic hind limb model, GFP expression increased from the 4th week and could be detected at 8 weeks post-injection. The rAAV vector had superior gene expressing activity. Eight weeks after gene transfer, the mean blood flow was significantly higher in the AAV-VEGF/BMP group. Orthotopic ossification was radiographically evident, and capillary growth and calcium deposits were obvious in this group.

Conclusion:

 
AAV-mediated VEGF and BMP gene transfer stimulates angiogenesis and bone regeneration and may be a new therapeutic technique for the treatment of avascular necrosis of the femoral head (ANFH).

Keywords:

adeno-associated virus; vascular endothelial growth factor; bone morphogenetic protein (BMP); avascular necrosis of the femoral head (ANFH); gene therapy
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Introduction

Recent insight into the pathogenesis of avascular necrosis of the femoral head (ANFH) has not identified satisfactory methods to increase blood circulation in necrotic areas of the femoral head, to promote bone regeneration, or to prevent osteonecrosis. The rapid development of gene therapy technology is increasingly recognized as a new therapeutic option for the treatment of ANFH, especially through therapeutic neovascularization and bone formation. Among growth factors, vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP) play important roles and have been extensively studied.
The VEGF family of growth factors is one of the most important cytokine families involved in angiogenesis. These factors promote the division of vascular endothelial cells and induce angiopoiesis. VEGF growth factors are essential for bone formation and repair during the bone regeneration process, which directly attracts endothelial cells and osteoclasts and enhances the differentiation of osteoblasts1, 2. BMP growth factors are the only signaling molecules that are individually sufficient for the induction of bone formation at orthotopic and heterotopic sites. They have defined roles in stimulating the proliferation and differentiation of mesenchymal and osteoprogenitor cells and have efficient bone induction activity3, 4. Because bone formation is a coordinated process involving the BMP and VEGF growth factors5, 6, orchestrating the timing with which these two factors are expressed may greatly enhance this process.
Choosing a safe and effective vector system to transfer and correctly express a target gene during gene therapy is important. Several different strategies have been examined for the delivery of genes of interest, including the use of naked DNA or an adenoviral vector. Treatment with naked DNA is simple and well tolerated by the recipient organism due to its low toxicity and weak induction of immune responses. However, the transduction efficiency is significantly lower when compared with other methods. The adenovirus has frequently been the vector of choice for gene transfer because it is able to transduce a variety of cells with high efciency. However, adenoviral vectors have major limitations, including a lack of sustained expression, the antigenicity of viral proteins that are targeted by both humoral immunity and cytotoxic T lymphocytes, and possible toxicity at high doses. However, there are many inherent features of the adeno-associated virus system that make it an attractive option as a human viral vector. AAV is a non-pathogenic, defective human parvovirus that requires the presence of a helper virus, such as adenovirus or herpes virus, for productive infection7, 8. Other advantages of this vector system include its low immunogenicity, its ability to transduce both dividing and non-dividing cells, the potential to integrate into specific sites, its ability to achieve long-term gene expression (even in vivo), and its broad tropism, allowing for the efficient transduction of diverse organs9. These features make AAV attractive and efficient for gene transfer in vitro and local injection in vivo.
To enhance neovascularization and bone regeneration during osteonecrosis therapy, we constructed adeno-associated viruses co-expressing hVEGF165 and hBMP-7 (rAAV-VEGF165-IRES-BMP-7) and detected their effect on gene expression and biological activity in vitro and in vivo. These data demonstrate the synergistic action of these two genes and may provide a new therapeutic option for ANFH.
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Materials and methods

Materials and reagents

The rAAV-hVEGF165-IRES-hBMP-7 (AAV-VEGF/BMP), rAAV-hVEGF165-GFP (AAV-VEGF), rAAV-hBMP-7-GFP (AAV-BMP), and rAAV-IRES-GFP (AAV-GFP) plasmids were constructed by Dr Xiang-hui HUANG. Human embryonic kidney cells-293 (HEK-293) and human umbilical vein endothelial cells (HUVECs) were obtained from the Department of Orthopedic Surgery in the Second Affiliated Hospital of Xi'an Jiaotong University. Male New Zealand rabbits (two months old, weighing 2.0–3.0 kg) were obtained from the experimental animal center of Xi'an Jiao Tong University. All animal protocols followed the recommendations and guidelines of the National Institutes of Health and were approved by the Xi'an Jiao Tong University Animal Care and Use Committee. The AAV helper-free system was obtained from Stratagene (La Jolla, CA, USA). A schematic representation of the structure of the plasmids in the AAV Helper Free System is provided in Figure 1A.
Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author(A) Schematic representation of the structure of plasmids in AAV Helper Free System. (B) Conceptual diagram of construction of pAAV-hVEGF165-IRES-hBMP-7. hVEGF165 gene (600 bp) and hBMP-7 gene (1300 bp) were respectively inserted into upstream MCS and downstream MCS located on either side of IRES sequence (631 bp). The length of the bicistronic frame is 2.5 kb.
Full figure and legend (72K)

rAAV vector production

The construction of the rAAV-hVEGF165-IRES-hBMP-7 (AAV-VEGF/BMP), rAAV-hVEGF165-GFP (AAV-VEGF), rAAV-hBMP-7-GFP (AAV-BMP), and rAAV-IRES-GFP (AAV-GFP) vectors was carried out as previously described10. The structure of the pAAV-hVEGF165-IRES-hBMP-7 vector is shown in Figure 1B. IRES sequences were incorporated into the pAAV MCS to construct a bicistronic vector with two multiple cloning sites. Then, the hVEGF165 (Pubmed NM-003376) and hBMP-7 (Pubmed NM-001719) genes were inserted into the upstream and downstream MCS, respectively. The length of the bicistronic frame is 2.5 kb, which is within the capacity of the vector. The AAV helper-free system was used to generate recombinant AAV. HEK-293 cells were cultured in H-DMEM supplemented with 10% fetal bovine serum containing 20 mg/mL penicillin-streptomycin and incubated with 5% CO2 at 37 oC. The AAV vector was co-transfected with the pAAV-helper and pAAV-RC vectors into HEK 293 cells by a calcium phosphate method according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). A primary virus stock was collected 72 h after transfection and further concentrated and purified by chloroform/PEG8000 protocols11. The recombinant adeno-associated virus had a titer of 5.5×1011 vp/mL.

Rabbit bone marrow-derived mesenchymal stem cells (BMSC) culture and rAAV infection in vitro

Male New Zealand rabbits were used to obtain rabbit BMSCs. The cells were harvested by gently flushing the tibiae and femora with L-DMEM. Density gradient centrifugation and adherent screening methods were used to isolate BMSCs as previously described12. The cells were cultured in L-DMEM supplemented with 10% fetal bovine serum containing 20 mg/mL penicillin-streptomycin and incubated with 5% CO2 at 37 oC. Following the 3rd passage, BMSCs (5×104 cells/well) were seeded onto 24-well plates 24 h before rAAV infection. By taking into account the cytopathogenic effect, infection efficiency, and cost of recombinant virus, we determined that the best multiplication of infection (MOI) for infecting rabbit BMSCs with rAAV was 5×104 vp/cell. The four rAAV virus variants were introduced into BMSCs using this MOI. Cells were incubated as above and were swirled gently at 30-min intervals. One hour later, the medium was replaced with L-DMEM supplemented with 10% fetal bovine serum. Medium was then completely replaced every three days.

Rabbit hind limb ischemia model and rAAV infection in vivo

Male New Zealand rabbits were kept under specific pathogen free conditions and supplied with sterile food and acidified water. The hind limb ischemia model was developed as described previously13. Rabbits were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). Under a surgical microscope, a vertical longitudinal incision was made in the right hind limb. The right femoral arteries were separated from the origin of the external iliac artery, ligated, and completely excised. Immediately after ligation of the femoral artery, the four rAAV virus variants were each injected into five different sites14 on the three major thigh muscles of each rabbit (5.5×1011 vp/20 μL per site), including the adductor (two sites), the quadriceps (two sites), and the semimembranous (one site) muscles. Subsequently, the skin was sutured. After surgery, all animals were housed under standard conditions (temperature: 21±1 °C; humidity: 55%–60%) with food and water continuously available. The hind limbs were mobilized without any fixation. To prevent infection, animals received prophylactic injections of gentamicin (0.03 mg·kg−1·d−1, im) within 3 days after surgery.
Rabbits were sacrificed at various time points post-injection to characterize gene expression efficiency and the effects on angiopoiesis and bone regeneration in vivo. Each group contained 30 rabbits and was divided into four experimental subgroups: group A (n=6) was examined at week 2 for GFP expression (n=3) and immunoblotting (n=3), group B (n=6) at week 4 for GFP expression (n=3) and immunoblotting (n=3), group C (n=9) at week 6 for GFP expression (n=3), immunoblotting (n=3), and ELISA (n=3), and group D (n=9) at week 8 for GFP expression (n=3) and for blood flow measurement, X-ray radiography, and immunohistochemistry (n=6).

Reporter gene (GFP) expression in vitro and in vivo

Following 3, 7, 14, and 28 days of infection with AAV-GFP virus in vitro, the expression of GFP protein was observed by inverted fluorescence microscopy. At 2, 4, 6, and 8 weeks post-injection in vivo, the muscles injected with the AAV-GFP virus were sliced by the frozen section method and the expression of the GFP protein was observed as above. Each assay was performed in triplicate.

Preparation of culture medium and assessment of VEGF165 and BMP-7 gene expression

Total cellular RNA was isolated at 1, 2, 3, 7, 14, 21, and 28 days following infection with the AAV-GFP, AAV-VEGF, AAV-BMP or AAV-VEGF/BMP viruses using TRIzol Reagent (Invitrogen). Extracted RNA was treated with DNase I (Takara, Tokyo, Japan) to eliminate DNA contamination, and first-strand cDNA was synthesized with random hexamer primers using the reverse first-strand cDNA synthesis kit from MBI Fermentas (Glen Burnie, MD, USA). PCR was performed to amplify humanVEGF165 (forward primer 5′-CCATCGATATGAACTTTCTGCTGTCTTG-3′; reverse primer 5′-CGGAATTCTCACCGCCTCGGCTTGTC-3′) and BMP-7 (forward primer 5′-GGCCGGATCCATGCACGTGCGCTCACTGCG-3′; reverse primer 5′-GGCCGTCGACCTAGTGGCAGCCACAG-3′). β-actin (forward primer 5′-GAGGGAAATCGTGCGTGAC-3′; reverse primer 5′-TAGGAGCCAGGGCAGTAATCT-3′) was detected by RT-PCR as an internal control. PCR was performed using the following program: 94 °C for 3 min for one cycle and 35 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s. The PCR products were electrophoresed on ethidium bromide-stained 2.0% agarose gels. Each assay was performed in triplicate.

Muscle extract preparation and assessment of VEGF165 and BMP-7 gene expression

At 2, 4, and 6 weeks following injection with the AAV-GFP, AAV-VEGF, AAV-BMP, or AAV-VEGF/BMP viruses, the frozen muscles were pulverized in liquid nitrogen and homogenized in 3 mL of ice-cold lysis buffer (1% Nonidet P-40; 50 mmol/L Tris-HCl, pH 7.4; 150 mmol/L NaCl; 200 U/mL aprotinin; 1 mmol/L phenylmethylsulfonyl fluoride, PMSF). The tissue lysates (50 mg of protein) were separated by 12% polyacrylamide gel electrophoresis and blotted onto polyvinylidene diuoride membranes. Immunoblotting was performed with anti-human VEGF165 and BMP-7 antibodies and the specic binding of the antibody was visualized with an ECL detection system. At 6 weeks post-injection, muscle extracts were measured with an enzyme-linked immunosorbent assay (ELISA) kit using the Biotrak ELISA system (R&D, Minneapolis, MN, USA) according to the manufacturer's instructions. Each assay was performed in triplicate.

Angiogenic and osteogenic in vitro assays

Tube formation assay
 
HUVECs were cultured as previously described15. Basement membrane matrigel matrix (BD, Bedford, MA, USA) was diluted by serum-free medium, added to a 24-well plate, and incubated at 37 °C for 30 min to allow solidification to occur. HUVECs (5×104 cells/well) were seeded on the matrigel and fresh L-DMEM medium supplemented with 10% FBS was added. Next, 1 mL of culture supernatant was harvested from the AAV-GFP, AAV-VEGF, AAV-BMP, or AAV-VEGF/BMP groups 14 days post-infection and added to the 24-well plate. The plate was then incubated at 37 oC with 5% CO2 for 12 h. The images of tube formation were captured under a light microscope from three random fields, and quantification of the tubes was analyzed by image processing software (Media Cybernetics, USA) to assess the biological activity of VEGF in vitro.
Mineralization assay
 
BMSCs were infected with the four virus groups above. The cells were then cultured in L-DMEM supplemented with 10% fetal bovine serum containing 20 mg/mL penicillin-streptomycin with 5% CO2 at 37 °C (the culture medium did not contain osteogenic induction factors, such as ascorbic acid, β-glycerophosphate, or dexamethasone). Mineralization effects were detected by von Kossa and alizarin red (AZR) staining16 for calcium deposits 4 weeks post-infection and observed using an inverted phase contrast microscope. The images of mineral nodules were captured under a light microscope from three random fields, and quantification of the mineral nodules was analyzed by image processing software to assess the biological activity of BMP in vitro.

Blood flow measurement and orthotopic bone formation in vivo

Eight weeks after injection, rabbits in the four groups were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). Blood flow in the anterior tibial artery of ischemic and normal hind limbs was measured at rest with an Aspen Advanced Doppler ultrasound device from Acuson (Siemens Medical Solutions, Mountain View, CA, USA) using a perivascular flow probe and calculated by the inlay automatic processing software. The data were expressed as a percentage of the contralateral limbs. Three separate measurements were performed for each rabbit at every time point and the results were averaged. In addition, rabbits in the four groups were subjected to X-ray radiography to assess orthotopic bone formation.

Histological assessment

Eight weeks after injection, thigh muscle tissue sections of ischemic limbs from the four groups were harvested and xed in 10% neutral-buffered formalin. To identify the proliferation of capillary endothelial cells, tissue sections were immunostained for CD34. The monoclonal antibody against CD34 was applied at a 1:500 dilution after blocking with 1% normal bovine serum. Subsequent incubation with biotinylated horse anti-mouse IgG and an ABC Elite kit (Santa Cruz) was performed. The number of CD34-positive vessels was counted at a magnification of 200×, and twenty fields from each typical slide were counted (mean number of capillaries per square millimeter). To assess orthotopic bone formation, the slides were stained by von Kossa staining to detect mineralization.

Statistical analysis

The results are reported as means±standard deviation. The normality of the data distribution was assessed with the Shapiro-Wilk (W) test. ANOVA followed by the Fisher's test was conducted to assess differences among treatment groups. Statistical significance was set at a P-value less than or equal to 0.05. The SPSS mathematical statistics software used for this analysis was purchased from SPSS Inc (version 8; SPSS Inc, Chicago, IL, USA).
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Results

Animal condition after rAAV infection

There were no symptoms of local or systemic toxicity after rAAV infection. In the region of the injection sites, no inflammatory reaction, such as rubeosis, engorgement, or abscessus, was observed. The activities of all animals were normal. There was no systemic toxicity, such as nutation, instability of gait, anhelation, retardation, cyanosis, or convulsion. No animals died before the end of the experiments.

GFP gene expression

In vitro: GFP protein expression could be detected on the third day post-infection. However, the efficiency and density of infection were unstable. The expression of GFP protein increased from the 7th day and could be detected at 28 days post-infection (Figures 2A, 2B). In vivo: With prolonged infection time, GFP protein expression increased from the 4th week and could be detected at 8 weeks post-infection (Figures 2C, 2D).
Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorRepresentative images of GFP protein expression. (A–B) on the 3rd, 7th, 14th, and 28th days after rAAV-IRES-GFP virus transfection in vitro. (A) Magnification×100; (B) Magnification×200; (C–D) on the 2nd, 4th, 6th, and 8th weeks after rAAV-IRES-GFP virus injection in vivo. (C) Magnification×100; (D) magnification×200.
Full figure and legend (161K)

Efficient genes expression of hVEGF165 and hBMP-7

To confirm hVEGF165 and hBMP-7 gene expression in vitro, RT-PCR assays were performed. As shown in Figure 3A–3D, the sizes of the PCR products for VEGF165, BMP-7, and β-actin were 600 bp, 1300 bp, and 340 bp, respectively. With prolonged infection time, the intensity of the VEGF165 and BMP-7 bands increased in the AAV-VEGF/BMP group. Together, these data demonstrate that VEGF165 was expressed in the AAV-VEGF and AAV-VEGF/BMP groups but not in the AAV-BMP and AAV-GFP groups and that BMP-7 was expressed in the AAV-BMP and AAV-VEGF/BMP groups but not in the AAV-VEGF and AAV-GFP groups. Protein expression of 2, 4, and 6 weeks following injection with AAV-VEGF/BMPin vivo is shown in Figure 3E–3H. Expression of the VEGF165 and BMP-7 proteins was visualized by Western blot analysis. Strong staining at the expected molecular weights of 23 kDa (hVEGF165), 55 kDa (hBMP-7), and 43 kDa (β-actin) was observed. With prolonged infection time, the intensity of the VEGF165 and BMP-7 bands increased. These data demonstrate that VEGF165 was expressed in the AAV-VEGF and AAV-VEGF/BMP groups but not in the AAV-BMP and AAV-GFP groups and that BMP-7 was expressed in the AAV-BMP and AAV-VEGF/BMP groups but not in the AAV-VEGF and AAV-GFP groups. As shown in Figure 3I, 3J, the production of hVEGF165 and hBMP-7 was quantified in relevant muscle extracts 6 weeks post-injection. The average amounts of hVEGF165 protein in the AAV-VEGF/BMP and AAV-VEGF groups were significantly higher than those in the AAV-GFP and AAV-BMP groups (P<0.05, n=30). The average amounts of hBMP-7 protein in the AAV-VEGF/BMP and AAV-BMP groups were significantly higher than those in the AAV-GFP and AAV-VEGF groups (P<0.05, n=30).
Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorExpression of hVEGF165 and hBMP-7. (A–D) Representative images of RT-PCR assay of AAV-GFP group (A), AAV-VEGF group (B), AAV-BMP group (C) and AAV-VEGF/BMP group (D). The size of the PCR products for hVEGF165, hBMP-7, and β-actin were 600 bp, 1300 bp, and 340 bp, respectively. With prolonged infection time, the brightness of the VEGF165 or BMP-7 bands increased in AAV-VEGF/BMP group. No hVEGF165 or hBMP-7 band could be detected in AAV-GFP group. Track 1–7 stands for the 1st, 2nd, 3rd, 7th, 14th, 21st, and 28th days post-transfection. (E–H) Representative images of Western blotting assay of AAV-GFP group (E), AAV-VEGF group (F), AAV-BMP group (G) and AAV-VEGF/BMP group (H). The molecular weights of hVEGF165, hBMP-7, and β-actin were 23 kDa, 55 kDa and 43 kDa respectively. Strong staining with the expected molecular weight was observed in AAV-VEGF/BMP group, and no hVEGF165 or hBMP-7 band was observed in AAV-GFP group. (I) ELISA assay for VEGF protein expression. The data is expressed as the mean±SD from three independent experiments. bP<0.05 vs AAV-GFP group, eP<0.05 vs AAV-BMP group. (J) ELISA assay for BMP protein expression. The data is expressed as the mean±SD from three independent experiments. bP<0.05 vs AAV-GFP group. eP<0.05 vs AAV-VEGF group.
Full figure and legend (53K)

Biological activity of hVEGF165 and hBMP-7 in vitro

As shown in Figure 4A, hVEGF165 secreted from BMSCs in the AAV-VEGF/BMP group enhanced HUVEC migration, proliferation, and tube formation in comparison with the other three groups. The number of tubes in the AAV-VEGF/BMP group was significantly higher than that in the AAV-GFP and AAV-BMP groups. However, there was no statistical difference between the AAV-VEGF/BMP group and the AAV-VEGF group (Figure 4B). In addition, the mineralization effect of hBMP-7 was detected by von Kossa (Figure 5A) and alizarin red staining (Figure 5B). The AAV-VEGF/BMP group displayed stronger osteogenic activity than all the other groups. The number of mineralized nodules in the AAV-VEGF/BMP group was significantly higher than that in the AAV-GFP and AAV-VEGF groups. However, there was no statistical difference between the AAV-VEGF/BMP group and the AAV-BMP group (Figure 5C).

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