The placenta theory and the origin of infantile hemangioma

The pathogenesis of infantile hemangioma is unknown. In recent years, much of the focus has been placed at identifying the cell type(s) responsible for tumor initiation. New discoveries in infantile hemangioma suggest an involvement of progenitor cells in the pathogenesis of this vascular tumor. Both embryonic and extra-embryonic tissues have been postulated as potential sources for these progenitor cells. This review focuses on the placental theory, which proposes that a fetal placental progenitor is the cell type of origin for infantile hemangioma. Special emphasis will be placed on placental vasculogenesis and the presence and transit of placental progenitor cells during gestation.     

Vascular morphogenesis by adult bone marrow progenitor cells in three-dimensional fibrin matrices

The neovascularization of tissues is accomplished by two distinct processes: de novo formation of blood vessels through the assembly of progenitor cells during early prenatal development (vasculogenesis), and expansion of a pre-existing vascular network by endothelial cell sprouting (angiogenesis), the main mechanism of blood vessel growth in postnatal life. Evidence exists that adult bone marrow (BM)-derived progenitor cells can contribute to the formation of new vessels by their incorporation into sites of active angiogenesis. Aim of this study was to investigate the in vitro self-organizing capacity of human BM mononuclear cells (BMMNC) to induce vascular morphogenesis in a three-dimensional (3D) matrix environment in the absence of pre-existing vessels. Whole BMMNC as well as the adherent and non-adherent fractions of BMMNC were embedded in fibrin gels and cultured for 3-4 weeks without additional growth factors. The expression of hematopoietic-, endothelial-, smooth muscle lineage, and stem cell markers was analyzed by immunohistochemistry and confocal laser-scanning microscopy. The culture of unselected BMMNC in 3D fibrin matrices led to the formation of cell clusters expressing the endothelial progenitor cell (EPC) markers CD133, CD34, vascular endothelial growth factor receptor (VEGFR)-2, and c-kit, with stellar shaped spreading of peripheral elongated cells forming tube-like structures with increasing complexity over time. Cluster formation was dependent on the presence of both adherent and non-adherent BMMNC without the requirement of external growth factors. Developed vascular structures expressed the endothelial markers CD34, VEGFR-2, CD31, von Willebrand Factor (vWF), and podocalyxin, showed basement-membrane-lined lumina containing CD45+ cells and were surrounded by alpha-smooth muscle actin (SMA) expressing mural cells. Our data demonstrate that adult human BM progenitor cells can induce a dynamic self organization process to create vascular structures within avascular 3D fibrin matrices suggesting a possible alternative mechanism of adult vascular development without involvement of pre-existing vascular structures.     

Endothelial progenitor cells for postnatal vasculogenesis

In the past decade, researchers have defined committed stem or progenitor cells from various tissues, including bone marrow, peripheral blood, brain, liver, and reproductive organs, in both adult animals and humans. Whereas most cells in adult organs are composed of differentiated cells, which express a variety of specific phenotypic genes adapted to each organ's environment, quiescent stem or progenitor cells are maintained locally or in the systemic circulation and are activated by environmental stimuli for physiological and pathological tissue regeneration. Recently, endothelial progenitor cells (EPCs) were isolated from peripheral blood CD34, Flk-1, or AC133 antigen-positive cells, which are considered to include a hematopoietic stem cell population, and were shown to be incorporated into foci of neovascularization. This finding, that circulating EPCs may home to sites of neovascularization and differentiate into endothelial cells in situ, is consistent with "vasculogenesis," a critical paradigm for embryonic neovascularization, and suggests that vasculogenesis and angiogenesis may constitute complementary mechanisms for postnatal neovascularization. Previous reports demonstrating therapeutic potential of EPC transplantation in animal models of hindlimb and myocardial ischemia opened the way to the clinical application of cell therapy: the replacement of diseased or degenerating cell populations, tissues, and organs. In this review, we summarize biological features of EPCs and speculate on the utility of EPCs for vascular and general medicine. cell transplantation; ischemia; neovascularization; stem cell     

Vascular wall resident progenitor cells: A review

The vessel wall has usually been thought to be relatively quiescent. But the discovery of progenitor cells in many tissues and in the vasculature itself has led to a reconsideration of the vascular biology. The presence of circulating endothelial and smooth muscle progenitors able to home to the injured vascular wall is a firm acquisition; less known is the notion, coming from embryonic and adult tissue studies, that stem cells able to differentiate into endothelial cells and smooth muscle cells also reside in the arterial wall. Moreover, the existence of a vasculogenic zone has recently been identified in adult human arteries; this niche-like zone is believed to act as a source of progenitors for postnatal vasculogenesis. From the literature it is already apparent that a complex interplay between circulating and resident vascular wall progenitors takes place during embryonal and postnatal life; a structural/functional disarray of these intimate stem cell compartments could hamper appropriate vascular repair, the development of vascular wall disease being the direct clinical consequence in adult life. This review gives an overview of adult large vessel progenitors established in the vascular wall during embryogenesis and their role in the maintenance of wall homeostasis.     

Mesenchymal stem cells and neovascularization: role of platelet-derived growth factor receptors

There is now accumulating evidence that bone marrow-derived mesenchymal stem cells (MSCs) make an important contribution to postnatal vasculogenesis, especially during tissue ischaemia and tumour vascularization. Identifying mechanisms which regulate the role of MSCs in vasculogenesis is a key therapeutic objective, since while increased neovascularization can be advantageous during tissue ischaemia, it is deleterious during tumourigenesis. The potent angiogenic stimulant vascular endothelial growth factor (VEGF) is known to regulate MSC mobilization and recruitment to sites of neovascularization, as well as directing the differentiation of MSCs to a vascular cell fate. Despite the fact that MSCs did not express VEGF receptors, we have recently identified that VEGF-A can stimulate platelet-derived growth factor (PDGF) receptors, which regulates MSC migration and proliferation. This review focuses on the role of PDGF receptors in regulating the vascular cell fate of MSCs, with emphasis on the function of the novel VEGF-A/PDGF receptor signalling mechanism.     

Vascular Endothelial Growth Factor (VEGF) Bioavailability Regulates Angiogenesis and Intestinal Stem and Progenitor Cell Proliferation during Postnatal Small Intestinal Development

Background

Vascular endothelial growth factor (VEGF) is a highly conserved, master regulatory molecule required for endothelial cell proliferation, organization, migration and branching morphogenesis. Podocoryne carnea and drosophila, which lack endothelial cells and a vascular system, express VEGF homologs, indicating potential roles beyond angiogenesis and vasculogenesis. The role of VEGF in the development and homeostasis of the postnatal small intestine is unknown. We hypothesized regulating VEGF bioavailability in the postnatal small intestine would exhibit effects beyond the vasculature and influence epithelial cell stem/progenitor populations.

Methods

VEGF mutant mice were created that overexpressed VEGF in the brush border of epithelium via the villin promotor following doxycycline treatment. To decrease VEGF bioavailability, sFlt-1 mutant mice were generated that overexpressed the soluble VEGF receptor sFlt-1 upon doxycycline administration in the intestinal epithelium. Mice were analyzed after 21 days of doxycycline administration.

Results

Increased VEGF expression was confirmed by RT-qPCR and ELISA in the intestine of the VEGF mutants compared to littermates. The VEGF mutant duodenum demonstrated increased angiogenesis and vascular leak as compared to littermate controls. The VEGF mutant duodenum revealed taller villi and increased Ki-67-positive cells in the transit-amplifying zone with reduced Lgr5 expression. The duodenum of sFlt-1 mutants revealed shorter villi and longer crypts with reduced proliferation in the transit-amplifying zone, reduced expression of Dll1, Bmp4 and VE-cadherin, and increased expression of Sox9 and EphB2.

Conclusions

Manipulating VEGF bioavailability leads to profound effects on not only the intestinal vasculature, but epithelial stem and progenitor cells in the intestinal crypt. Elucidation of the crosstalk between VEGF signaling in the vasculature, mesenchyme and epithelial stem/progenitor cell populations may direct future cell therapies for intestinal dysfunction or disease.     

Precancerous stem cells can serve as tumor vasculogenic progenitors

Tumor neo-vascularization is critical for tumor growth, invasion and metastasis, which has been considered to be mediated by a mechanism of angiogenesis. However, histopathological studies have suggested that tumor cells might be the progenitor for tumor vasculature. Recently, we have reported that the precancerous stem cells (pCSCs) representing the early stage of developing cancer stem cells (CSCs), have the potential for both benign and malignant differentiation. Therefore, we investigated whether pCSCs serve as progenitors for tumor vasculogenesis. Herein, we report that in the pCSC-derived tumors, most blood vessels were derived from pCSCs. Some pCSCs constitutively expressed vasculogenic receptor VEGFR-2, which can be up-regulated by hypoxia and angiogenesis-promoting cytokines, such as GM-CSF, Flt3 ligand, and IL-13. The pCSCs are much more potent in tumor vasculogenesis than the differentiated tumor monocytic cells (TMCs) from the same tumor, which had comparable or even higher capacity to produce some vascular growth factors, suggesting that the potent tumor vasculogenesis of pCSCs is associated with their intrinsic stem-like property. Consistently tumor vasculogenesis was also observed in human cancers such as cervical cancer and breast cancer and xenograft lymphoma. Our studies indicate that pCSCs can serve as tumor vasculogenic stem/progenitor cells (TVPCs), and may explain why anti-angiogenic cancer therapy trials are facing challenge.     

β-adrenergic receptor antagonists inhibit vasculogenesis of embryonic stem cells by downregulation of nitric oxide generation and interference with VEGF signalling

The β-adrenoceptor antagonist Propranolol has been successfully used to treat infantile hemangioma. However, its mechanism of action is so far unknown. The hypothesis of this research was that β-adrenoceptor antagonists may interfere with endothelial cell differentiation of stem cells. Specifically, the effects of the non-specific β-adrenergic receptor (β-adrenoceptor) antagonist Propranolol, the β₁-adrenoceptor-specific antagonist Atenolol and the β₂-adrenoceptor-specific antagonist ICI118,551 on vasculogenesis of mouse embryonic stem (ES) cells were investigated. All three β-blockers dose-dependently downregulated formation of capillary structures in ES cell-derived embryoid bodies and decreased the expression of the vascular cell markers CD31 and VE-cadherin. Furthermore, β-blockers downregulated the expression of fibroblast growth factor-2 (FGF-2), hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor 165 (VEGF₁₆₅), VEGF receptor 2 (VEGF-R2) and phospho VEGF-R2, as well as neuropilin 1 (NRP1) and plexin-B1 which are essential modulators of embryonic angiogenesis with additional roles in vessel remodelling and arteriogenesis. Under conditions of β-adrenoceptor inhibition, the endogenous generation of nitric oxide (NO) as well as the phosphorylation of endothelial nitric oxide synthase (eNOS) was decreased in embryoid bodies, whereas an increase in NO generation was observed with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP). Consequently, vasculogenesis of ES cells was restored upon treatment of differentiating ES cells with β-adrenoceptor antagonists in the presence of NO donor. In summary, our data suggest that β-blockers impair vasculogenesis of ES cells by interfering with NO generation which could be the explanation for their anti-angiogenic effects in infantile hemangioma.     

Increased circulating AC133+ CD34+ endothelial progenitor cells in children with hemangioma

Hemangioma is the most common soft-tissue tumor of infancy. Despite the frequency of these vascular tumors, the origin of hemangioma-endothelial cells is unknown. Circulating endothelial progenitor cells (EPCs) have recently been identified as vascular stem cells with the capacity to contribute to postnatal vascular development. We have attempted to determine whether circulating EPCs are increased in hemangioma patients and thereby provide insight into the role of EPCs in hemangioma growth. METHODS AND RESULTS: Peripheral blood mononuclear cells (PBMCs) were isolated from hemangioma patients undergoing surgical resection (N = 5) and from age-matched controls (N = 5) undergoing strabismus correction surgery. PBMCs were stained with fluorescent-labeled antibodies for AC133, CD34, and VEGFR2/KDR. Fluorescent-labeled isotype antibodies served as negative controls. Histologic sections of surgical specimens were stained with the specific hemangioma markers Glut1, CD32, and merosin, to confirm the diagnosis of common hemangioma of infancy. EPCs harvested from healthy adult volunteers were stained with Glut1, CD32, and merosin, to assess whether cultured EPCs express known hemangioma markers. Hemangioma patients had a 15-fold increase in the number of circulating CD34 AC133 dual-staining cells relative to controls (0.78+/-0.14% vs.0.052+/-0.017%, respectively). Similarly, the number of PBMCs that stained positively for both CD34 and KDR was also increased in hemangioma patients (0.49+/-0.074% vs. 0.19+/-0.041% in controls). Cultured EPCs stained positively for the known hemangioma markers Glut1, CD32, merosin. CONCLUSIONS: This is the first study to suggest a role for EPCs in the pathogenesis of hemangioma. Our results imply that increased levels of circulating EPCs may contribute to the formation of this vascular tumor.     

Therapeutic myocardial angiogenesis with vascular endothelial growth factors

Emerging evidence has shown that administration of angiogenic growth factors, either as recombinant protein or by gene transfer, can augment tissue perfusion through neovascularization in animal models of myocardial and hindlimb ischemia. Many cytokines have angiogenic activity; one of those that have been best studied in animal models and clinical trials is vascular endothelial growth factor (VEGF). VEGF has been known to be a key regulator of physiologic and pathologic angiogenesis associated with tumor. Recently the effect of VEGF is not restricted to the direct angiogenic effect in vivo but includes mobilization of bone-marrow-derived endothelial progenitor cells and augmentation of postnatal vasculogenesis in situ. Clinical trials of therapeutic angiogenesis with VEGF in patients with end-stage coronary artery disease have shown increases in exercise time and reductions in anginal symptoms and have provided objective evidence of improved perfusion and left ventricular function. Larger scale placebo-controlled trials with recombinant protein (rhVEGF165) have been limited to intracoronary and intravenous administration and have shown favorable trends in exercise time and angina frequency. Small-scale, placebo-controlled, randomized clinical trials of gene transfer (phVEGF-2) via thoracotomy or percutaneous intramyocardial delivery demonstrated significant improvement of both subjective symptoms and objective measures of myocardial ischemia. Both therapeutic modalities appear to be safe and well tolerated. Further studies are required to determine the optimal dose, formulation, route of administration, and combinations of growth factors and the utility of adjunctive endothelial progenitor cell or other stem cell supplementation, to provide safe and effective therapeutic myocardial neovascularization.     

Neovascularization and Hematopoietic Stem Cells

Vasculogenesis and angiogenesis are the major forms of blood vessel formation. Angiogenesis is the process where new vessels grow from pre-existing blood vessels, and is very important in the functional recovery of pathological conditions, such as wound healing and ischemic heart diseases. The development of better animal model and imaging technologies in past decades has greatly enriched our understanding on vasculogenesis and angiogenesis processes. Hypoxia turned out to be an important driving force for angiogenesis in various ischemic conditions. It stimulates expression of many growth factors like vascular endothelial growth factor, platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor, which play critical role in induction of angiogenesis. Other cellular components like monocytes, T cells, neutrophils, and platelets also play significant role in induction and regulation of angiogenesis. Various stem/progenitor cells also being recruited to the ischemic sites play crucial role in the angiogenesis process. Pre-clinical studies showed that stem/progenitor cells with/without combination of growth factors induce neovascularization in the ischemic tissues in various animal models. In this review, we will discuss about the fundamental factors that regulate the angiogenesis process and the use of stem cells as therapeutic regime for the treatment of ischemic diseases.     

Placental Growth Factor Expression Is Required for Bone Marrow Endothelial Cell Support of Primitive Murine Hematopoietic Cells

Two distinct microenvironmental niches that regulate hematopoietic stem/progenitor cell physiology in the adult bone marrow have been proposed; the endosteal and the vascular niche. While extensive studies have been performed relating to molecular interactions in the endosteal niche, the mechanisms that regulate hematopoietic stem/progenitor cell interaction with bone marrow endothelial cells are less well defined. Here we demonstrate that endothelial cells derived from the bone marrow supported hematopoietic stem/progenitor cells to a higher degree than other endothelial or stromal cell populations. This support was dependant upon placental growth factor expression, as genetic knockdown of mRNA levels reduced the ability of endothelial cells to support hematopoietic stem/progenitor cells in vitro. Furthermore, using an in vivo model of recovery from radiation induced myelosuppression, we demonstrate that bone marrow endothelial cells were able to augment the recovery of the hematopoietic stem/progenitor cells. However, this effect was diminished when the same cells with reduced placental growth factor expression were administered, possibly owing to a reduced homing of the cells to the bone marrow vasculature. Our data suggest that placental growth factor elaborated from bone marrow endothelial cells mediates the regulatory effects of the vascular niche on hematopoietic stem/progenitor cell physiology.     

Hypoxia-induced pulmonary artery adventitial remodeling and neovascularization: contribution of progenitor cells

Information is rapidly emerging regarding the important role of the arterial vasa vasorum in a variety of systemic vascular diseases. In addition, increasing evidence suggests that progenitor cells of bone marrow (BM) origin may contribute to postnatal neovascularization and/or vascular wall thickening that is characteristic in some forms of systemic vascular disease. Little is known regarding postnatal vasa formation and the role of BM-derived progenitor cells in the setting of pulmonary hypertension (PH). We sought to determine the effects of chronic hypoxia on the density of vasa vasorum in the pulmonary artery and to evaluate if BM-derived progenitor cells contribute to the increased vessel wall mass in a bovine model of hypoxia-induced PH. Quantitative morphometric analyses of lung tissue from normoxic and hypoxic calves revealed that hypoxia results in a dramatic expansion of the pulmonary artery adventitial vasa vasorum. Flow cytometric analysis demonstrated that cells expressing the transmembrane tyrosine kinase receptor for stem cell factor, c-kit, are mobilized from the BM in the circulation in response to hypoxia. Immunohistochemistry revealed an increase in the expression of c-kit+ cells together with vascular endothelial growth factor, fibronectin, and thrombin in the hypoxia-induced remodeled pulmonary artery vessel wall. Circulating mononuclear cells isolated from neonatal calves exposed to hypoxia were found to differentiate into endothelial and smooth muscle cell phenotypes depending on culture conditions. From these observations, we suggest that the vasa vasorum and circulating progenitor cells could be involved in vessel wall thickening in the setting of hypoxia-induced PH.     

Ras signaling directs endothelial specification of VEGFR2+ vascular progenitor cells

Vascular endothelial growth factor receptor 2 (VEGFR2) transmits signals of crucial importance to vasculogenesis, including proliferation, migration, and differentiation of vascular progenitor cells. Embryonic stem cell-derived VEGFR2(+) mesodermal cells differentiate into mural lineage in the presence of platelet derived growth factor (PDGF)-BB or serum but into endothelial lineage in response to VEGF-A. We found that inhibition of H-Ras function by a farnesyltransferase inhibitor or a knockdown technique results in selective suppression of VEGF-A-induced endothelial specification. Experiments with ex vivo whole-embryo culture as well as analysis of H-ras(-/-) mice also supported this conclusion. Furthermore, expression of a constitutively active H-Ras[G12V] in VEGFR2(+) progenitor cells resulted in endothelial differentiation through the extracellular signal-related kinase (Erk) pathway. Both VEGF-A and PDGF-BB activated Ras in VEGFR2(+) progenitor cells 5 min after treatment. However, VEGF-A, but not PDGF-BB, activated Ras 6-9 h after treatment, preceding the induction of endothelial markers. VEGF-A thus activates temporally distinct Ras-Erk signaling to direct endothelial specification of VEGFR2(+) vascular progenitor cells.     

Calcium/calmodulin-dependent protein kinase II activity regulates the proliferative potential of growth plate chondrocytes

For tissues that develop throughout embryogenesis and into postnatal life, the generation of differentiated cells to promote tissue growth is at odds with the requirement to maintain the stem cell/progenitor cell population to preserve future growth potential. In the growth plate cartilage, this balance is achieved in part by establishing a proliferative phase that amplifies the number of progenitor cells prior to terminal differentiation into hypertrophic chondrocytes. Here, we show that endogenous calcium/calmodulin-dependent protein kinase II (CamkII, also known as Camk2) activity is upregulated prior to hypertrophy and that loss of CamkII function substantially blocks the transition from proliferation to hypertrophy. Wnt signaling and Pthrp-induced phosphatase activity negatively regulate CamkII activity. Release of this repression results in activation of multiple effector pathways, including Runx2- and β-catenin-dependent pathways. We present an integrated model for the regulation of proliferation potential by CamkII activity that has important implications for studies of growth control and adult progenitor/stem cell populations.     

Bone marrow subsets differentiate into endothelial cells and pericytes contributing to Ewing's tumor vessels

Hematopoietic progenitor cells arising from bone marrow (BM) are known to contribute to the formation and expansion of tumor vasculature. However, whether different subsets of these cells have different roles in this process is unclear. To investigate the roles of BM-derived progenitor cell subpopulations in the formation of tumor vasculature in a Ewing's sarcoma model, we used a functional assay based on endothelial cell and pericyte differentiation in vivo. Fluorescence-activated cell sorting of human cord blood/BM or mouse BM from green fluorescent protein transgenic mice was used to isolate human CD34+/CD38(-), CD34+/CD45+, and CD34(-)/CD45+ cells and mouse Sca1+/Gr1+, Sca1(-)/Gr1+, VEGFR1+, and VEGFR2+ cells. Each of these progenitor subpopulations was separately injected intravenously into nude mice bearing Ewing's sarcoma tumors. Tumors were resected 1 week later and analyzed using immunohistochemistry and confocal microscopy for the presence of migrated progenitor cells expressing endothelial, pericyte, or inflammatory cell surface markers. We showed two distinct patterns of stem cell infiltration. Human CD34+/CD45+ and CD34+/CD38(-) and murine VEGFR2+ and Sca1+/Gr1+ cells migrated to Ewing's tumors, colocalized with the tumor vascular network, and differentiated into cells expressing either endothelial markers (mouse CD31 or human vascular endothelial cadherin) or the pericyte markers desmin and alpha-smooth muscle actin. By contrast, human CD34(-)/CD45+ and mouse Sca1(-)/Gr1+ cells migrated predominantly to sites outside of the tumor vasculature and differentiated into monocytes/macrophages expressing F4/80 or CD14. Our data indicate that only specific BM stem/progenitor subpopulations participate in Ewing's sarcoma tumor vasculogenesis.     

The role of multipotential stem cells in human infantile hemangioma

Hemangioma is the most common benign vascular tumor in infants and children with unknown etiology and pathogenesis. It is characterized by rapid proliferation followed by a slow involution phase. Histological analyses of infantile hemangioma (IH) in the early proliferating phase have generated a number of developmental theories suggesting an embryonic or primitive cell origin. We here hypothesize the IH may originate from multipotential stem cells. Further investigations of these hemangioma-initiating cells may improve our understanding of their function and possibly lead to novel therapeutic modalities for hemangiomas.     

Adult neural stem cells: response to stroke injury and potential for therapeutic applications

The plasticity of neural stem/progenitor cells allows a variety of different responses to many environmental cues. In the past decade, significant research has gone into understanding the regulation of neural stem/progenitor cell properties, because of their promise for cell replacement therapies in adult neurological diseases. Both endogenous and grafted neural stem/progenitor cells are known to have the ability to migrate long distances to lesioned sites after brain injury and differentiate into new neurons. Several chemokines and growth factors, including stromal cell-derived factor-1 and vascular endothelial growth factor, have been shown to stimulate the proliferation, differentiation, and migration of neural stem/progenitor cells, and investigators have now begun to identify the critical downstream effectors and signaling mechanisms that regulate these processes. Both our own lab and others have shown that the extracellular matrix and matrix remodeling factors play a critical role in directing cell differentiation and migration of adult neural stem/progenitor cells within injured sites. Identification of these and other molecular pathways involved in stem cell homing into ischemic areas is vital for the development of new treatments. To ensure the best functional recovery, regenerative therapy may require the application of a combination approach that includes cell replacement, trophic support, and neural protection. Here we review the current state of our knowledge about endogenous adult and exogenous neural stem/progenitor cells as potential therapeutic agents for central nervous system injuries.     

Embryonic development of the proepicardium and coronary vessels

In the last few years, an increasing interest in progenitor cells has been noted. These cells are a source of undifferentiated elements from which cellular components of tissues and organs develop. Such progenitor tissue delivering stem cells for cardiac development is the proepicardium. The proepicardium is a transient organ which occurs near the venous pole of the embryonic heart and protrudes to the pericardial cavity. The proepicardium is a source of the epicardial epithelium delivering cellular components of vascular wall and interstitial tissue fibroblasts. It contributes partially to a fibrous tissue skeleton of the heart. Epicardial derived cells play also an inductive role in differentiation of cardiac myocytes into conductive tissue of the heart. Coronary vessel formation proceeds by vasculogenesis and angiogenesis. The first tubules are formed from blood islands which subsequently coalesce forming the primitive vascular plexus. Coronary arteries are formed by directional growth of vascular protrusions towards the aorta and establishing contact with the aortic wall. The coronary vascular wall matures by attaching smooth muscle cell precursors and fibroblast precursors to the endothelial cell wall. The cells of tunica media differentiate subsequently into vascular smooth muscle by acquiring specific contractile and cytoskeletal markers of smooth muscle cells in a proximal - distal direction. The coronary artery wall matures first before cardiac veins. Maturity of the vessel wall is demonstrated by the specific shape of the internal surface of the vascular wall.