Induction of angiogenesis by bovine brain derived class 1 heparin-binding growth factor

The angiogenic capacity of the class 1 heparin-binding growth factor from bovine brain, an anionic endothelial cell mitogen of Mr 16 000, has been evaluated. Its ability to induce the growth of new blood vessels has been assessed by means of two established assay systems. On the embryonic chick chorioallantoic membrane dose-response studies demonstrate that 160 ng (10 pmol) of mitogen is required to induce angiogenesis in greater than 50% of the eggs within 72 h. In the presence of 1 unit of exogenous heparin only 40 ng of mitogen (2.5 pmol) is needed to induce a similar response. Moreover, this occurs within 48 h, indicating that heparin also augments the angiogenic response by enhancing the rate of induction of angiogenesis. Eighty nanograms (5 pmol) of mitogen also induces the ingrowth of new blood vessels into the rabbit cornea, both in the presence and in the absence of heparin. These results establish that the class 1 heparin-binding growth factor from bovine brain is an angiogenesis factor. Importantly, the neovascularization induced by this angiogenesis factor is enhanced by heparin. The mechanistic implications for neovascularization under certain normal and pathological conditions are discussed.     

Identification of smooth muscle-derived foam cells in the atherosclerotic plaque of human aorta with monoclonal antibody IIG10

We have developed a monoclonal antibody that specifically interacts with a surface antigen of human fibroblasts and smooth muscle cells. The antibody (antibody IIG10) recognizes a polypeptide of molecular mass 330,000, revealed by immunoblotting in fibroblast and smooth muscle cell extract, but not in vascular endothelial cells, peritoneal macrophages, peripheral blood lymphocytes nor hepatocytes. In tissue sections the antibody stained smooth muscle cells of myometrium, aorta and smaller blood vessels, and fibroblasts of connective tissue. Specificity of the antibody was further confirmed by double staining of aorta sections. Antibody IIG10 was used to identify smooth muscle-derived foam cells in the atherosclerotic plaque of human aorta.     

New insights into saccular development and vascular formation in lung allografts under the renal capsule

The study of distal lung morphogenesis and vascular development would be greatly facilitated by an in vitro or ex vivo experimental model. In this study we show that the growth of mouse embryonic day 12.5 lung rudiments implanted underneath the kidney capsules of syngeneic or immunodeficient hosts follows closely lung development in utero. The epithelium develops extensively with both proximal and distal differentiation to the saccular stage. The vasculature also develops extensively. Large blood vessels accompany large airways and capillaries develop within the saccular walls. Interestingly, vessels in the lung grafts develop from endothelial progenitor cells endogenous to the explants and host vessels do not vascularize the grafts independently. This suggests that embryonic lungs possess mechanisms to prevent the inappropriate ingrowth of surrounding vessels. However, vessels in the lung grafts do connect to host vessels, showing that embryonic lungs have the ability to stimulate host angiogenesis and recruit host vessel connections. These data support the hypothesis that the lung vasculature develops by both vasculogenic and angiogenic processes: a vascular network develops in situ in lung mesenchyme, which is then connected to angiogenic processes from central vessels. The lung renal capsule allograft is thus an excellent model to study the development of the pulmonary vasculature and of late fetal lung development that requires a functional blood supply.     

In vitro bone formation using muscle-derived cells: a new paradigm for bone tissue engineering using polymer-bone morphogenetic protein matrices

Over 800,000 bone grafting procedures are performed in the United States annually, creating a demand for viable alternatives to autogenous bone, the grafting standard in osseous repair. The objective of this study was to examine the efficacy of a BMP-polymer matrix in inducing the expression of the osteoblastic phenotype and in vitro bone formation by muscle-derived cells. Specifically, we evaluated the ability of bone morphogenetic protein-7 (BMP-7), delivered from a poly(lactide-co-glycolide) (PLAGA) matrix, to induce the differentiation of cells derived from rabbit skeletal muscle into osteoblast-like cells and subsequently form mineralized tissue. Results confirmed that muscle-derived cells attached and proliferated on the PLAGA substrates. BMP-7 released from PLAGA induced the muscle-derived cells to increase bone marker expression and form mineralized cultures. These results demonstrate the efficacy of a BMP-polymer matrix in inducing the expression of the osteoblastic phenotype by muscle-derived cells and present a new paradigm for bone tissue engineering.     

Vascular development in early human embryos and in teratomas derived from human embryonic stem cells

During early human embryonic development, blood vessels are stimulated to grow, branch, and invade developing tissues and organs. Pluripotent human embryonic stem cells (hESCs) are endowed with the capacity to differentiate into cells of blood and lymphatic vessels. The present study aimed to follow vasculogenesis during the early stages of developing human vasculature and to examine whether human neovasculogenesis within teratomas generated in SCID mice from hESCs follows a similar course and can be used as a model for the development of human vasculature. Markers and gene profiling of smooth muscle cells and endothelial cells of blood and lymphatic vessels were used to follow neovasculogenesis and lymphangiogenesis in early developing human embryos (4-8 weeks) and in teratomas generated from hESCs. The involvement of vascular smooth muscle cells in the early stages of developing human embryonic blood vessels is demonstrated, as well as the remodeling kinetics of the developing human embryonic blood and lymphatic vasculature. In teratomas, human vascular cells were demonstrated to be associated with developing blood vessels. Processes of intensive remodeling of blood vessels during the early stages of human development are indicated by the upregulation of angiogenic factors and specific structural proteins. At the same time, evidence for lymphatic sprouting and moderate activation of lymphangiogenesis is demonstrated during these developmental stages. In the teratomas induced by hESCs, human angiogenesis and lymphangiogenesis are relatively insignificant. The main source of blood vessels developing within the teratomas is provided by the murine host. We conclude that the teratoma model has only limited value as a model to study human neovasculogenesis and that other in vitro methods for spontaneous and guided differentiation of hESCs may prove more useful.     

Heparinized collagen scaffolds with and without growth factors for the repair of diaphragmatic hernia

A regenerative medicine approach to restore the morphology and function of the diaphragm in congenital diaphragmatic hernia is especially challenging because of the position and flat nature of this organ, allowing cell ingrowth primarily from the perimeter. Use of porous collagen scaffolds for the closure of surgically created diaphragmatic defects in rats has been shown feasible, but better ingrowth of cells, specifically blood vessels and muscle cells, is warranted. To stimulate this process, heparin, a glycosaminoglycan involved in growth factor binding, was covalently bound to porous collagenous scaffolds (14%), with or without vascular endothelial growth factor (VEGF; 0.4 µg/mg scaffold), hepatocyte growth factor (HGF; 0.5 µg/mg scaffold) or a combination of VEGF + HGF (0.2 + 0.5 µg/mg scaffold). All components were located primarily at the outside of scaffolds. Scaffolds were implanted in the diaphragm of rats and evaluated after 2 and 12 weeks. No herniations or eventrations were observed, and in several cases, growth factor-substituted scaffolds showed macroscopically visible blood vessels at the lung site. The addition of heparin led to an accelerated ingrowth of blood vessels at 2 weeks. In all scaffold types, giant cells and immune cells were present primarily at the liver side of the scaffold, and immune cells and individual macrophages at the lung side; these cell types decreased in number from week 2 to week 12. The addition of growth factors did not influence cellular response to the scaffolds, indicating that further optimization with respect to dosage and release profile is needed.     

ACE2 and AT4R are present in diseased human blood vessels

A growing body of evidence suggests that the angiotensin II fragments, Ang(1-7) and Ang(3-8), have a vasoactive role, however ACE2, the enzyme that produces Ang(1-7), or AT4R, the receptor that binds Ang (3-8), have yet been simultaneously localised in both normal and diseased human conduit blood vessels. We sought to determine the immunohistochemical distribution of ACE2 and the AT4R in human internal mammary and radial arteries from patients undergoing coronary artery bypass surgery. We found that ACE2 positive cells were abundant in both normal and diseased vessels, being present in neo-intima and in media. ACE2 positive immunoreactivity was not present in the endothelial layer of the conduit vessels, but was clearly evident in small newly formed angiogenic vessels as well as the vaso vasorum. Endothelial AT4R immunoreactivity were rarely observed in either normal and diseased arteries, but AT4R positive cells were observed adjacent to the internal elastic lamine in the internal mammary artery, in the neo-intima of radial arteries, as well as in the media of both internal mammary artery and radial artery. AT4R was abundant in vaso vasorum and within small angiogenic vessels. Both AT4R and ACE2 co-localised with smooth muscle cell alpha actin. This study identifies smooth muscle cell alpha actin positive ACE2 and AT4R in human blood vessels as well as in angiogenic vessels, indicating a possible role for these enzymes in pathological disease.     

Survival in vitro of motoneurons identified or purified by novel antibody-based methods is selectively enhanced by muscle-derived factors

Motoneurons were identified in vitro by a new method using the SC1 monoclonal antibody. They constituted up to 30% of total neurons in cultures of whole spinal cord from 4.5-day chicken embryos, and survived for at least 5 days in the presence of muscle extract, but not in its absence. By contrast, other neurons and floor-plate cells survived without muscle-derived factors. Motoneurons were purified to homogeneity by 'panning' on dishes coated with SC1 antibody; they developed rapidly even in the absence of other spinal cells. Concentrations of muscle extract required for half-maximal motoneuron survival were indistinguishable in pure and mixed cultures, suggesting that muscle-derived factors act directly on motoneurons. Other purified growth factors tested, including ciliary neurotrophic factor, did not have the survival-promoting activity of muscle.     

Histological and immunocytochemical investigation of human coronary vessel development with anti-CD34 antibodies

Information about embryonic development of coronary endothelium is the main clue for the creation of new methods in tissue engineering for treatment of ischemic heart diseases. The purpose of the research was to describe human coronary vessels development on early stages of the prenatal ontogenesis. The first step in human coronary vessels development is the formation of endothelium de novo by transformation of some epicardial and, possibly, endocardial cells. The next step is the ingrowth of sinus venosus endothelium in subepicardium over ventricles and atria, which gives rise to the coronary vessels. Only after 7 days does the primitive coronary plexus of the heart communicate with aorta (third step). During this period, some subepicardial vessels invade myocardium and some intramyocardial vessels contact with the heart cavity. Such intercommunications could help in regulation of blood circulation in primitive coronary plexus before establishment of effective contacts between arterial and venous vessels—excess of blood could be discharged directly into the heart cavity. Additional population of CD34+ cells were revealed inside condensed mesenchyme of the conotruncus; it participates in the formation of vasa vasorum in the aorta. Epicardium and sinus venosus generate endothelium of coronary vessels by neovasculo- and angiogenesis, respectively. During a week after ingrowth of vessels from SV and before their ingrowth to the aorta, ventriculo-coronary communications could be found in the heart.     

Muscle-derived stem cells isolated as non-adherent population give rise to cardiac, skeletal muscle and neural lineages

Stem cells with the ability to differentiate in specialized cell types can be extracted from a wide array of adult tissues including skeletal muscle. Here we have analyzed a population of cells isolated from skeletal muscle on the basis of their poor adherence on uncoated or collagen-coated dishes that show multi-lineage differentiation in vitro. When analysed under proliferative conditions, these cells express stem cell surface markers Sca-1 (65%) and Bcrp-1 (80%) but also MyoD (15%), Neuronal beta III-tubulin (25%), GFAP (30%) or Nkx2.5 (1%). Although capable of growing as non-attached spheres for months, when given an appropriate matrix, these cells adhere giving rise to skeletal muscle, neuronal and cardiac muscle cell lineages. A similar cell population could not be isolated from either bone marrow or cardiac tissue suggesting their specificity to skeletal muscle. When injected into damaged muscle, these non-adherent muscle-derived cells are retrieved expressing Pax7, in a sublaminar position characterizing satellite cells and participate in forming new myofibers. These data show that a non-adherent stem cell population can be specifically isolated and expanded from skeletal muscle and upon attachment to a matrix spontaneously differentiate into muscle, cardiac and neuronal lineages in vitro. Although competing with resident satellite cells, these cells are shown to significantly contribute to repair of injured muscle in vivo supporting that a similar muscle-derived non-adherent cell population from human muscle may be useful in treatment of neuromuscular disorders.     

Tumstatin, the NC1 domain of alpha3 chain of type IV collagen, is an endogenous inhibitor of pathological angiogenesis and suppresses tumor growth

Angiogenesis, the formation of new blood vessels, is required for physiological development of vertebrates and repair of damaged tissue, but in the pathological setting contributes to progression of cancer. During tumor growth, angiogenesis is supported by up-regulation of angiogenic stimulators (pro-angiogenic) and down-regulation of angiogenic inhibitors (anti-angiogenic). The switch to the angiogenic phenotype (angiogenic switch) allows the tumors to grow and facilitate metastasis. The bioactive NC1 domain of type IV collagen alpha3 chain, called tumstatin, imparts anti-tumor activity by inducing apoptosis of proliferating endothelial cells. Tumstatin binds to alphaVbeta3 integrin via a mechanism independent of the RGD-sequence recognition and inhibits cap-dependent protein synthesis in the proliferating endothelial cells. The physiological level of tumstatin is controlled by matrix metalloproteinase-9, which most effectively cleaves it from the basement membrane and its physiological concentration in the circulation keeps pathological angiogenesis and tumor growth in check. These findings suggest that tumstatin functions as an endogenous inhibitor of pathological angiogenesis and functions as a novel suppressor of proliferating endothelial cells and growth of tumors.     

Inhibition of angiogenesis by the antineoplastic agents mitoxantrone and bisantrene

The effects of mitoxantrone and bisantrene on angiogenic responses induced by tumor cell-conditioned media in the avascular cornea of rat eye have been evaluated. Both mitoxantrone and bisantrene effectively inhibited, in a concentration-dependent manner, angiogenesis induced by conditioned media obtained from either a hamster buccal pouch carcinoma cell line or P388D1 murine macrophage-like cells. Whereas vessel ingrowth in corneas containing tumor cell-conditioned media was detected as early as day 2 or 3 and was maximal by day 7, inclusion of mitoxantrone or bisantrene in the conditioned media at a 1:1 ratio (160 microM mitoxantrone or 32 microM bisantrene) resulted in complete inhibition of angiogenesis throughout the 14-day evaluation period. When concentrations of 64 and 32 microM mitoxantrone or 13 and 6.4 microM bisantrene were employed there was a marked delay in the appearance of capillary blood vessels (day 5 to 7) and a reduction in the intensity of angiogenic responses. No untoward toxicity to the tissue was observed at the concentrations of mitoxantrone or bisantrene employed.     

Angiogenic synergism,vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF-2

The establishment of functional and stable vascular networks is essential for angiogenic therapy. Here we report that a combination of two angiogenic factors, platelet-derived growth factor (PDGF)-BB and fibroblast growth factor (FGF)-2, synergistically induces vascular networks, which remain stable for more than a year even after depletion of angiogenic factors. In both rat and rabbit ischemic hind limb models, PDGF-BB and FGF-2 together markedly stimulated collateral arteriogenesis after ligation of the femoral artery, with a significant increase in vascularization and improvement in paw blood flow. A possible mechanism of angiogenic synergism between PDGF-BB and FGF-2 involves upregulation of the expression of PDGF receptor (PDGFR)-alpha and PDGFR-beta by FGF-2 in newly formed blood vessels. Our data show that a specific combination of angiogenic factors establishes functional and stable vascular networks, and provides guidance for the ongoing clinical trials of angiogenic factors for the treatment of ischemic diseases.     

Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing

Several recent studies suggest the isolation of stem cells in skeletal muscle, but the functional properties of these muscle-derived stem cells is still unclear. In the present study, we report the purification of muscle-derived stem cells from the mdx mouse, an animal model for Duchenne muscular dystrophy. We show that enrichment of desmin(+) cells using the preplate technique from mouse primary muscle cell culture also enriches a cell population expressing CD34 and Bcl-2. The CD34(+) cells and Bcl-2(+) cells were found to reside within the basal lamina, where satellite cells are normally found. Clonal isolation and characterization from this CD34(+)Bcl-2(+) enriched population yielded a putative muscle-derived stem cell, mc13, that is capable of differentiating into both myogenic and osteogenic lineage in vitro and in vivo. The mc13 cells are c-kit and CD45 negative and express: desmin, c-met and MNF, three markers expressed in early myogenic progenitors; Flk-1, a mouse homologue of KDR recently identified in humans as a key marker in hematopoietic cells with stem cell-like characteristics; and Sca-1, a marker for both skeletal muscle and hematopoietic stem cells. Intramuscular, and more importantly, intravenous injection of mc13 cells result in muscle regeneration and partial restoration of dystrophin in mdx mice. Transplantation of mc13 cells engineered to secrete osteogenic protein differentiate in osteogenic lineage and accelerate healing of a skull defect in SCID mice. Taken together, these results suggest the isolation of a population of muscle-derived stem cells capable of improving both muscle regeneration and bone healing.     

Generation of a leukotriene-like substance from porcine vascular and other tissues

When chopped porcine vascular tissue was incubated with A23187 in the presence of a cyclo-oxygenase inhibitor, a substance with the biological properties of a leukotriene was released into the supernatant. The highest concentrations of leukotriene-like material were released by coronary and pulmonary arteries and the adventitia removed from these vessels, the greatest amount being released by the adventitia. Smaller quantities of leukotriene-like substance were released from other blood vessels but not from aorta, atrial or ventricular smooth muscle. Release of the leukotriene-like material was time-dependent. The activity of the leukotriene-like substance was antagonised by FPL 55712 and its release was inhibited by BW 755c. Leukotriene-like material was also generated from guinea-pig, rat, rabbit and dog blood vessels incubated with A23187 and from guinea-pig vessels during antigen challenge.     

Generation of a leukotriene-like substance from porcine vascular and other tissues

When chopped porcine vascular tissue was incubated with A23187 in the presence of a cyclo-oxygenase inhibitor, a substance with the biological properties of a leukotriene was released into the supernatant. The highest concentrations of leukotriene-like material were released by coronary and pulmonary arteries and the adventitia removed from these vessels, the greatest amount being released by the adventitia. Smaller quantities of leukotriene-like substance were released from other blood vessels but not from aorta, atrial or venticular smooth muscle. Release of the leukotriene-like material was time-dependent. The activity of the leukotriene-like substance was antagonised by FPL 55712 and its release was inhibited by BW 755c. Leukotriene-like material was also generated from guinea-pig, rat, rabbit and dog blood vessels incubated with A23187 and from guinea-pig vessels during antigen challenge.     

Responses of vascular smooth muscle cell to extracellular matrix degradation.

Vessels remodel to compensate for increases in blood flow/pressure. The chronic exposure of blood vessels to increased flow and circulatory redox-homocysteine may injure vascular endothelium and disrupt elastic laminae. In order to understand the role of extracellular matrix (ECM) degradation in vascular structure and function, we isolated human vascular smooth muscle cells (VSMC) from normal and injured coronary arteries. The apparently normal vessels were isolated from explanted human hearts. The vessels were injured by inserting a blade into the lumen of the vessel, which damages the inner elastic laminae in the vessel wall and polarizes the VSMC by producing a pseudopodial phenotypic shift in VSMC. This shift is characteristic of migratory, invasive, and contractile nature of VSMC. We measured extracellular matrix metalloproteinases (MMPs), tissue plasminogen activator (tPA), tissue inhibitor of metalloproteinase (TIMP), and collagen I expression in VSMC by specific substrate zymography and Northern blot analyses. The injured and elastin peptide, val-gly-val-ala-pro-gly, treated VSMC synthesized active MMPs and reduced expression of TIMP. The level of tPA and collagen type I was induced in the injured, invasive VSMC and in the val-gly-val-ala-pro-gly treated cells. To demonstrate the angiogenic role of elastin peptide to VSMC we performed in vitro organ culture with rings from normal coronary artery. After 3 days in culture the vascular rings in the collagen gel containing elastin peptide elaborated MMP activity and sprouted and grew. The results suggest that val-gly-val-ala-pro-gly peptide generated at the site of proteolysis during vascular injury may have angiogenic activity.     

Isolation of muscle derived stem cells from rat and its smooth muscle differentiation [corrected

We investigated whether stem cells (MDSC) from primary cultures of rat skeletal muscle can differentiate into the smooth muscle lineage in response to vascular endothelial growth factor (VEGF) and coculture with bladder smooth muscle cells. The MDSC were isolated from gastrocnemius muscle biopsies of normal 3-6 week-old Sprague-Dawley rats and purified by the preplate technique. Cells that took approximately 6 days to adhere to the collagen-coated flasks were termed late preplate (LP) cells, and were used in all the experiments. The early plate (EP) cells (pp1-pp4) contained some myogenic cells but were mostly fibroblasts (< 15% desmin+ cells) whereas the LP cells (pp5-pp6) were highly purified muscle-derived cells (pp6) (> 90% desmin+ cells). The muscle-derived stem cells (LP cells) were CD34+ or Sca-1+, CD45- and desmin+ by immunohistochemical staining. After two days of co-culture with bladder smooth muscle cells, about 25% of the muscle-derived stem cells were positive for alpha-smooth muscle actin (alpha-SMA)+. RT-PCR for alpha-SMA was positive in the VEGF stimulated MDSC, but negative in the absence of VEGF. In conclusion, rat muscle-derived stem cells exhibited stem cell properties (CD34+ or Sca-1+), and were not of hematogeous (CD45-) but of myogenic origin (desmin+). RT-PCR of alpha-SMA was positive in the VEGF stimulated muscle-derived stem cells.