Origin of coronary endothelial cells from epicardial mesothelium in avian embryos

It has been established that coronary vessels develop through self-assembly of mesenchymal vascular progenitors in the subepicardium. Mesenchymal precursors of vascular smooth muscle cells and fibroblasts are known to originate from an epithelial-to-mesenchymal transformation of the epicardial mesothelium, but the origin of the coronary endothelium is still obscure. We herein report that at least part of the population of the precursors of the coronary endothelium are epicardially-derived cells (EPDCs). We have performed an EPDC lineage study through retroviral and fluorescent labelling of the proepicardial and epicardial mesothelium of avian embryos. In all the experiments onlythe surface mesothelium was labelled after 3 h of reincubation. However, endothelial cells from subepicardial vessels were labelled after 24-48 h and endothelial cells of intramyocardial vessels were also labelled after 48-96 h of reincubation. In addition, the development of the coronary vessels was studied in quail-chick chimeras, obtaining results which also support a mesothelial origin for endothelial and smooth muscle cells. Finally, quail proepicardial explants cultured on Matrigel showed colocalization of cytokeratin and QH1 (mesothelial and endothelial markers, respectively) after 24 h. These results, taken together, suggest that EPDC show similar competence to that displayed by bipotential vascular progenitor cells [Yamashita et al., Nature 408: 92-96 (2000)] which are able to differentiate into endothelium or smooth muscle depending on their exposure to VEGF or PDGF-BB. It is conceivable that the earliest EPDC differentiate into endothelial cells in response to myocardially-secreted VEGF, while further EPDC would be recruited by the nascent capillaries via PDGFR-beta signalling, giving rise to mural cells.     

The dynamic expression of tenascin-C and tenascin-X during early heart development in the mouse

One of a family of extracellular matrix proteins, tenascin-C (TNC) is expressed in a spatiotemporally restricted pattern associated with tissue remodeling during embryonic development, wound healing, cancer invasion and tissue regeneration. Another form, tenascin-X (TNX), is found in most tissues but most predominantly in heart and muscle, often complementarily to TNC. The present analysis demonstrated their expression during early heart development, using mouse lines containing the lacZ gene targeted to the TNC locus, by RT-PCR, immunohistochemistry, and in situ hybridization. TNC was transiently expressed at important steps during heart development: (1) precardiac mesodermal cells differentiating to cardiomyocytes and endocardial cells at E 7.5 - 8.5; (2) cardiomyocytes in the outflow tract at E 8.5 - 12; (3) endocardial cells forming cushion tissue at E 9.5 - 13; and (4) mesenchymal cells in the proepicardial organ (PEO), the precursors of coronary vessels, at E 9.5. When PEO cells were transferred onto the heart surface, the expression of TNC was downregulated, while TNX was upregulated at E 11. Initially, epicardial cells around the AV groove and atrium started to express TNX. TNX-positive cells then gradually spread all over the entire surface of the heart and invaded and formed primitive vascular channels in the myocardium. Despite restricted expression at important sites and steps during cardiogenesis, the hearts of TNC deficient mice developed normally. No difference in the expression pattern of TNX were observed in TNC knockout and wild mice. These results suggest; (1) TNC could play important roles in the differentiation of cardiomyocytes and the early morphogenesis of the heart; (2) TNX could be involved in coronary vasculogenesis; (3) TNX does not compensate for the loss of TNC.     

Avian coronary endothelium is a mosaic of sinus venosus‐ and ventricle‐derived endothelial cells in a region‐specific manner

The origin of coronary endothelial cells (ECs) has been investigated in avian species, and the results showed that the coronary ECs originate from the proepicardial organ (PEO) and developing epicardium. Genetic approaches in mouse models showed that the major source of coronary ECs is the sinus venosus endothelium or ventricular endocardium. To clarify and reconcile the differences between avian and mouse species, we examined the source of coronary ECs in avian embryonic hearts. Using an enhanced green fluorescent protein‐Tol2 system and fluorescent dye labeling, four types of quail‐chick chimeras were made and quail‐specific endothelial marker (QH1) immunohistochemistry was performed. The developing PEO consisted of at least two cellular populations in origin, one was sinus venosus endothelium‐derived inner cells and the other was surface mesothelium‐derived cells. The majority of ECs in the coronary stems, ventricular free wall, and dorsal ventricular septum originated from the sinus venosus endothelium. The ventricular endocardium contributed mainly to the septal artery and a few cells to the coronary stems. Surface mesothelial cells of the PEO differentiated mainly into a smooth muscle phenotype, but a few differentiated into ECs. In avian species, the coronary endothelium had a heterogeneous origin in a region‐specific manner, and the sources of ECs were basically the same as those observed in mice.     

Tenascin-C enhances crosstalk signaling of integrin αvβ3/PDGFR-β complex by SRC recruitment promoting PDGF-induced proliferation and migration in smooth muscle cells

Migration and proliferation of smooth muscle cells (SMCs) are key events during neointimal formation in pathological conditions of vessels. Tenascin-C (TNC) is upregulated in the developing neointima of lesions. We evaluated the effects of TNC on responses of SMCs against platelet-derived growth factor (PDGF) stimulation. TNC coated on substrate promoted PDGF-BB-induced proliferation and migration of rat SMC cell line A10 in BrdU incorporation and transwell assays, respectively. Immunoblotting showed that TNC substrate enhanced autophosphorylation of PDGFR-β after PDGF-BB stimulation. Integrin αvβ3 is known to be a receptor for TNC in SMCs. In immunofluorescence and immunoblot of integrin αv subunit, clustering of αv-positive focal adhesions and upregulated αv expression were observed in the cells on TNC substrate. Immunoprecipitation using anti-integrin αvβ3 antibody demonstrated that PDGFR-β and integrin αvβ3 were co-precipitated and that the relative amount of PDGFR-β after the stimulation was increased by TNC treatment. TNC also promoted phosphorylation of focal adhesion kinase (FAK) at tyrosine (Y) 397 and Y925. The phosphorylated FAK was localized at focal adhesions in immunofluorescence. Phosphorylated SRC at Y418 was also seen at focal adhesions. Immunoprecipitation with αv antibody showed increased SRC association with the integrin signaling complex in the cells on TNC after PDGF treatment. In the cells on TNC substrate, crosstalk signaling between integrin αvβ3 and PDGFR-β could be amplified by SRC and FAK recruited to focal adhesions, followed by enhanced proliferation and migration of A10 cells by PDGF-BB.     

New phenotypic aspects of the decidual spiral artery wall during early post-implantation mouse pregnancy

During pregnancy the walls of decidual spiral arteries (SAs) undergo clinically important structural modifications crucial for embryo survival/growth and maternal health. However, the mechanisms of SA remodeling (SAR) are poorly understood. Although an important prerequisite to this understanding is knowledge about the phenotype of SA muscular wall prior to and during the beginning of mouse SAR, this remains largely unexplored and was the main aim of this work. Using histological and immunohistochemical techniques, this study shows for the first time that during early mouse gestation, from embryonic day 7.5 (E7.5) to E10.5, the decidual SA muscular coat is not a homogeneous structure, but consists of two concentric layers. The first is a largely one cell-thick sub-endothelial layer of contractile mural cells (positive for α-smooth muscle actin, calponin and SM22α) with pericyte characteristics (NG2 positive). The second layer is thicker, and evidence is presented that it may be of the synthetic/proliferative smooth muscle phenotype, based on absence (α-smooth muscle actin and calponin) or weak (SM22α) expression of contractile mural cell markers, and presence of synthetic smooth muscle characteristics (expression of non-muscle Myosin heavy chain-IIA and of the cell proliferation marker PCNA). Importantly, immunohistochemistry and morphometrics showed that the contractile mural cell layer although prominent at E7.5-E8.5, becomes drastically reduced by E10.5 and is undetectable by E12.5. In conclusion, this study reveals novel aspects of the decidual SA muscular coat phenotype prior to and during early SAR that may have important implications for understanding the mechanisms of SAR.     

Homocysteine is positively associated with cytokine IL-18 plasma levels in coronary artery bypass surgery patients

Homocysteine, cytokines (IL-18, IL-6, IL-8) are involved in vascular inflammation and coronary artery disease. Homocysteine influences endothelial IL-6 and IL-8 cytokine expression and release, however, an association between homocysteine and IL-18 has not been previously investigated in endothelial/smooth muscle cells and or in coronary artery disease. We report in 9 coronary artery bypass surgery (CABG) patients a positive correlation r = 0.86 between homocysteine and IL-18 plasma levels (p < 0.05). Plasma IL-18 levels are significantly higher in those patients with elevated homocysteine compared to those with normal levels (p < 0.02; 153 +/- 19 pg/ml versus 116 +/- 14 pg/ml respectively). Our in vitro cell culture studies suggest that the source of IL-18 in CABG patients with elevated homocysteine is not from vascular smooth muscle or endothelial cells.     

Variations in transfection efficiency of VEGF165 and VEGF121-cDNA

Little is known about the expression pattern of vascular endothelial growth factor (VEGF) among smooth muscle cells of different arterial regions. Therefore, we have conducted studies aimed at increasing expression of VEGF in cultured human smooth muscle cells (SMCs) from different sites: aorta, umbilical artery, and coronary artery. Two plasmids harboring human VEGF₁₂₁ and VEGF₁₆₅ isoforms, respectively, were constructed and lipotransfected into vascular SMCs, using the Fu-GENE 6. Extensive optimization of transfection conditions were performed prior to this. Different basal levels of VEGF were observed between cell types: from 0.51–0.95 pg/mL/μg protein in umbilical artery, through 2.32–2.39 pg/mL/μg protein in coronary artery, to 5.45–7.52 pg/mL/μg protein in aortic SMCs. Significant differences in responses to transfection were also observed: The increase in VEGF production was most pronounced in umbilical artery SMCs (e.g., with 4 μg VEGF₁₂₁-cDNA/in the wells)—an approximate 600-fold as opposed to an 18-fold increase in aortic SMCs and a 29-fold increase in coronary artery SMCs. In addition, we observed significant increases in proliferation rate of aortic and coronary endothelial cells (ECs), after incubation with conditioned medium from VEGF-transfected SMCs. Observed changes differed in relation to cell origin and isoform.     

Actin isoform and α1B-adrenoceptor gene expression in aortic and coronary smooth muscle is influenced by cyclical stretch

Summary The occurrence of vascular domains with specific biological and pharmacological characteristics suggests that smooth muscle cells in different arteries may respond differentially to a wide range of environmental stimuli. To determine if some of these vessel-specific differences may be attributable to mechano-sensitive gene regulation, the influence of cyclical stretch on the expression of actin isoform and α_1B-adrenoceptor genes was examined in aortic and coronary smooth muscle cells. Cells were seeded on an elastin substrate and subjected to maximal stretching (24% elongation) and relaxation cycles at a frequency of 120 cycles/min in a Flexercell strain unit for 72 h. Total RNA was extracted and hybridized to radiolabeled cDNA probes to assess gene expression. Stretch caused a greater reduction of actin isoform mRNA levels in aortic smooth muscle cells as compared to cells from the coronary artery. Steady-state mRNA levels of α_1B -adrenoceptor were also decreased by cyclical stretch in both cell types but the magnitude of the response was greater in coronary smooth muscle cells. No changes in α_1B-adrenoceptor or β/γ-actin steady-state mRNA levels were observed in H4IIE cells, a nonvascular, immortalized cell line. The relative gene expression of heat shock protein 70 was not influenced by the cyclic stretch regimen in any of these cell types. These results suggest that stretch may participate in the regulation of gene expression in vascular smooth muscle cells and that this response exhibits some degree of cell-specificity.     

Msx genes define a population of mural cell precursors required for head blood vessel maturation

Vessels are primarily formed from an inner endothelial layer that is secondarily covered by mural cells, namely vascular smooth muscle cells (VSMCs) in arteries and veins and pericytes in capillaries and veinules. We previously showed that, in the mouse embryo, Msx1(lacZ) and Msx2(lacZ) are expressed in mural cells and in a few endothelial cells. To unravel the role of Msx genes in vascular development, we have inactivated the two Msx genes specifically in mural cells by combining the Msx1(lacZ), Msx2(lox) and Sm22α-Cre alleles. Optical projection tomography demonstrated abnormal branching of the cephalic vessels in E11.5 mutant embryos. The carotid and vertebral arteries showed an increase in caliber that was related to reduced vascular smooth muscle coverage. Taking advantage of a newly constructed Msx1(CreERT2) allele, we demonstrated by lineage tracing that the primary defect lies in a population of VSMC precursors. The abnormal phenotype that ensues is a consequence of impaired BMP signaling in the VSMC precursors that leads to downregulation of the metalloprotease 2 (Mmp2) and Mmp9 genes, which are essential for cell migration and integration into the mural layer. Improper coverage by VSMCs secondarily leads to incomplete maturation of the endothelial layer. Our results demonstrate that both Msx1 and Msx2 are required for the recruitment of a population of neural crest-derived VSMCs.     

Arterial identity of endothelial cells is controlled by local cues.

The ephrins and their Eph receptors comprise the largest family of receptor tyrosine kinases. Studies on mice have revealed an important function of ephrin-B2 and Eph-B4 for the development of the arterial and venous vasculature, respectively, but the mechanisms regulating their expression have not been studied yet. We have cloned a chick ephrin-B2 cDNA probe. Expression was observed in endothelial cells of extra- and intraembryonic arteries and arterioles in all embryos studied from day 2 (stage 10 HH, before perfusion of the vessels) to day 16. Additionally, expression was found in the somites and neural tube in early stages, and later also in the smooth muscle cells of the aorta, parts of the Müllerian duct, dosal neural tube, and joints of the limbs. We isolated endothelial cells from the internal carotid artery and the vena cava of 14-day-old quail embryos and grafted them separately into day-3 chick embryos. Reincubation was performed until day 6 and the quail endothelial cells were identified with the QH1 antibody. The grafted arterial and venous endothelial cells expressed ephrin-B2 when they integrated into the lining of arteries. Cells that were not integrated into vessels, or into vessels other than arteries, were ephrin-B2-negative. The studies show that the expression of the arterial marker ephrin-B2 is controlled by local cues in arterial vessels of older embryos. Physical forces or the media smooth muscle cells may be involved in this process.     

Characterization of Pdgfrb-Cre transgenic mice reveals reduction of ROSA26 reporter activity in remodeling arteries

With the intention to modulate gene expression in vascular mural cells of remodeling vessels, we generated and characterized transgenic mouse lines with Cre recombinase under the control of the platelet-derived growth factor receptor-β promoter, referred to as Tg(Pdgfrb-Cre)(35Vli) . Transgenic mice were crossed with the Gt(ROSA)26Sor(tm1Sor) strain and examined for Cre activation by β-galactosidase activity, which was compared with endogenous Pdgfrb expression. In addition, Pdgfrb-Cre mice were used to drive expression of a conditional myc-tagged Cthrc1 transgene. There was good overlap of β-galactosidase activity with endogenous Pdgfrb immunoreactivity. However, dedifferentiation of vascular mural cells induced by carotid artery ligation revealed a dramatic discrepancy between ROSA26 reporter activity and Pdgfrb promoter driven Cre dependent myc-tagged Cthrc1 transgene expression. Our studies demonstrate the capability of the Pdgfrb-Cre mouse to drive conditional transgene expression as a result of prior Cre-mediated recombination in tissues known to express endogenous Pdgfrb. In addition, the study shows that ROSA26 promoter driven reporter mice are not suitable for lineage marking of smooth muscle in remodeling blood vessels.     

Neuron-like differentiation of adipose tissue-derived stromal cells and vascular smooth muscle cells

Adipose tissue-derived stromal cells (ADSC) have previously been shown to possess stem cell properties such as transdifferentiation and self-renewal. Because future clinical applications are likely to use these adult stem cells in an autologous fashion, we wished to establish and characterize rat ADSC for pre-clinical tests. In the present study, we showed that rat ADSC expressed stem cell markers CD34 and STRO-1 at passage 1 but only STRO-1 at passage 3. These cells could also be induced to differentiate into adipocytes, smooth muscle cells, and neuron-like cells, the latter of which expressed neuronal markers S100, nestin, and NF70. Isobutylmethylxanthine (IBMX), indomethacin (INDO), and insulin were the active ingredients in a previously established neural induction medium (NIM); however, here we showed that IBMX alone was as effective as NIM in the induction of morphological changes as well as neuronal marker expression. Finally, we showed that vascular smooth muscle cells could also be induced by either NIM or IBMX to differentiate into neuron-like cells that expressed NF70.     

Fibronectin promotes differentiation of neural crest progenitors endowed with smooth muscle cell potential

The neural crest (NC) is a model system used to investigate multipotency during vertebrate development. Environmental factors control NC cell fate decisions. Despite the well-known influence of extracellular matrix molecules in NC cell migration, the issue of whether they also influence NC cell differentiation has not been addressed at the single cell level. By analyzing mass and clonal cultures of mouse cephalic and quail trunk NC cells, we show for the first time that fibronectin (FN) promotes differentiation into the smooth muscle cell phenotype without affecting differentiation into glia, neurons, and melanocytes. Time course analysis indicated that the FN-induced effect was not related to massive cell death or proliferation of smooth muscle cells. Finally, by comparing clonal cultures of quail trunk NC cells grown on FN and collagen type IV (CLIV), we found that FN strongly increased both NC cell survival and the proportion of unipotent and oligopotent NC progenitors endowed with smooth muscle potential. In contrast, melanocytic progenitors were prominent in clonogenic NC cells grown on CLIV. Taken together, these results show that FN promotes NC cell differentiation along the smooth muscle lineage, and therefore plays an important role in fate decisions of NC progenitor cells.     

Changes in the Anatomic and Microscopic Structure and the Expression of HIF-1α and VEGF of the Yak Heart with Aging and Hypoxia

The study aimed to identify the changes of anatomic and microscopic structure and the expression and localization of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) in the myocardium and coronary artery of the yak heart adapted to chronic hypoxia with aging. Thirty-two yaks (1 day, 6 months, 1 year, 2 years, and 5 year old) were included, and immunoelectronmicroscopy, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) were used. Right ventricular hypertrophy was not present in yaks with aging. There was no intima thickening phenomenon in the coronary artery. The ultrastructure of myofibrils, mitochondria, and collagen fibers and the diameter and quantity of collagen changed significantly with aging. The enzymatic activity of complexes I, II, and V increased with age. Immunogold labeling showed the localization of HIF-1α protein in the cytoplasm and nuclei of endothelial cells and cytoplasm of cardiac muscle cells, and VEGF protein in the nuclei and perinuclei areas of smooth muscle cells of coronary artery, and in the cytoplasm and nuclei of endothelial cells. ELISA results showed that HIF-1α secretion significantly increased in the myocardium and coronary artery from an age of 1 day to 2 years of yaks and decreased in old yaks. However, VEGF protein always increased with aging. The findings of this study suggest that 6 months is a key age of yak before which there are some adaptive changes to deal with low-oxygen environment, and there is a maturation of the yak heart from the age of 6 months to 2 years.     

Microvascular and macrovascular endothelial cells produce different constrictor substances.

The media from cultured microvascular and macrovascular endothelial cells (conditioned media, CM) were collected and tested for constrictor activity in sheep coronary artery rings and tracheal smooth muscle strips in vitro (isometric force), expressed as percentage of contraction produced by 80 mM KCl. Both microvascular (micro) and macrovascular (macro) CM caused a sustained slow-onset contraction (P less than 0.05) of the coronary artery rings by 71 +/- 10% (micro; n = 7) and 67 +/- 8% (macro; n = 6) and tracheal smooth muscle strips by 33 +/- 14% (micro; n = 6) and 34 +/- 6% (macro; n = 11); the calcium antagonist gallopamil (10(-7) M) attenuated these effects by 25-55%. Unconditioned medium and medium conditioned by cultured tracheal smooth muscle cells had no constrictor activity on coronary artery rings or tracheal smooth muscle strips. Synthetic endothelin (ET-1) also produced contraction of coronary artery rings and tracheal smooth muscle strips. The mean levels of ET-1 measured by radioimmunoassay were 1,200 pg/ml in the macro CM and 33 pg/ml in the micro CM. Depleting macro CM of ET-1 by affinity columns constructed with protein A agarose and anti-ET-1 antibody removed the contractile activity for coronary artery rings and tracheal smooth muscle strips. Thus ET-1 did not appear to be the contractile substance in the micro CM. Preliminary characterization of the contractile substance in micro CM revealed that it was heat stable, had a molecular weight of less than 10,000, was inactivated by trypsin, and retained its activity after two cycles of freeze-thawing.(ABSTRACT TRUNCATED AT 250 WORDS)