Transforming growth factor beta responsiveness is modulated by the extracellular collagen matrix during hepatic ito cell culture

Hepatic sinusoidal Ito cells have the capacity to produce interstitial collagen types I and III as well as other matrix proteins and may be involved in hepatic fibrogenesis. Transforming growth factor beta (TGF beta) responsiveness was evaluated during in vitro cell culture, since increasing evidence suggests that this ubiquitous polypeptide can stimulate the production of collagenous proteins in a variety of cell types. TGF beta induced marked inhibition of Ito cell proliferation for cells grown on either a type I or a type IV collagen matrix. In marked contrast, the collagen synthetic response was considerably different for cells grown on a type I versus a type IV collagen matrix. When cells were grown on a type I collagen matrix, TGF beta caused a significant increase in the accumulation of collagen type I and III. When Ito cells were grown on a type IV collagen matrix, there was no stimulation of collagen production. TGF beta responsiveness was also evaluated in the setting of altered vitamin A concentrations. Freshly isolated Ito cells are engorged with vitamin A, the usual physiologic storage site for hepatic vitamin A. During in vitro culture and during in vivo fibrogenesis, Ito cells lose their vitamin A stores coincident with a transformation to a collagen-producing myofibroblast-like cell. When cultured Ito cells were grown on a type I collagen matrix and re-exposed to an increased concentration of vitamin A, the production of interstitial collagen was reduced. However, when the vitamin A-enriched Ito cells were exposed to TGF beta, the production of interstitial collagen was increased, similar to cells that had not received vitamin A.     

Extracellular matrix organization modulates fibroblast growth and growth factor responsiveness

To learn more about the relationship between extracellular matrix organization, cell shape, and cell growth control, we studied DNA synthesis by fibroblasts in collagen gels that were either attached to culture dishes or floating in culture medium during gel contraction. After 4 days of contraction, the collagen density (initially 1.5 mg/ml) reached 22 mg/ml in attached gels and 55 mg/ml in floating gels. After contraction, attached collagen gels were well organized; collagen fibrils were aligned in the plane of cell spreading; and fibroblasts had an elongated, bipolar morphology. Floating collagen gels, however, were unorganized; collagen fibrils were arranged randomly; and fibroblasts had a stellate morphology. DNA synthesis by fibroblasts in contracted collagen gels was suppressed if the gels were floating in medium but not if the gels were attached, and inhibition was independent of the extent of gel contraction. Therefore, growth of fibroblasts in contracted collagen gels could be regulated by differences in extracellular matrix organization and cell shape independently of extracellular matrix density. We also compared the responses of fibroblasts in contracted collagen gels and monolayer culture to peptide growth factors including fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, and interleukin 1. Cells in floating collagen gels were generally unresponsive to any of the growth factors. Cells in attached collagen gels and monolayer culture were affected similarly by fibroblast growth factor but not by the others. Our results indicate that extracellular matrix organization influenced not only cell growth, but also fibroblast responsiveness to peptide growth factors.     

Transforming growth factor type beta 1 modulates the effects of basic fibroblast growth factor on growth and phenotypic expression of rat astroblasts in vitro

In a search of the growth factors possibly involved in brain ontogenesis we have examined the effects of transforming growth factor beta 1 (TGF-beta 1) on the growth and phenotypic expression of rat astroblasts in primary culture. Along TGF-beta 1 elicited only a slight negative effect on the growth of these cells. However, this factor was found to modulate the mitogenic effects of other growth factors. On quiescent cells it potentiates the mitogenic effect of basic fibroblast growth factor (bFGF) but not that of other growth factors, namely, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and thrombin. TGF-beta 1 did not modulate significantly the stimulatory effect of these growth factors on the activity of the enzyme glutamine synthetase (GS); but kinetic studies showed that TGF-beta 1 delays the stimulation of GS activity. DNA synthesis monitored by the incorporation of [125I]iododeoxyuridine (125I-dUrd) was maximum after 24-30 h of treatment with bFGF. With bFGF plus TGF-beta 1 the maximum was shifted to 30-36 h. This shift is compatible with the idea that TGF-beta 1 induces responsiveness in some cells which are otherwise unresponsive to the mitogenic action of bFGF, and that this induction requires some time. This hypothesis is sustained by the observation that in cells treated for only 12 h with bFGF, the treatment with TGF-beta 1 for the same 12 h or for longer time did not stimulate significantly the cell growth. Stimulation occurred only when the bFGF treatment was continued after 12 h. Potentiation of the mitogenic effect of bFGF and shift of the maximum 125I-dUrd incorporation towards 24 h was seen with cells pretreated with TGF-beta 1. This potentiation effect decreased with increasing time between the two treatments. The potentiation effect of TGF-beta 1 is not mediated by an induction of new bFGF membrane receptors as seen by binding studies.     

Transforming growth factor beta1 (TGF-beta1) promotes endothelial cell survival during in vitro angiogenesis via an autocrine mechanism implicating TGF-alpha signaling

Mouse capillary endothelial cells (1G11 cell line) embedded in type I collagen gels undergo in vitro angiogenesis. Cells rapidly reorganize and form capillary-like structures when stimulated with serum. Transforming growth factor beta1 (TGF-beta1) alone can substitute for serum and induce cell survival and tubular network formation. This TGF-beta1-mediated angiogenic activity depends on phosphatidylinositol 3-kinase (PI3K) and p42/p44 mitogen-activated protein kinase (MAPK) signaling. We showed that specific inhibitors of either pathway (wortmannin, LY-294002, and PD-98059) all suppressed TGF-beta1-induced angiogenesis mainly by compromising cell survival. We established that TGF-beta1 stimulated the expression of TGF-alpha mRNA and protein, the tyrosine phosphorylation of a 170-kDa membrane protein representing the epidermal growth factor (EGF) receptor, and the delayed activation of PI3K/Akt and p42/p44 MAPK. Moreover, we showed that all these TGF-beta1-mediated signaling events, including tubular network formation, were suppressed by incubating TGF-beta1-stimulated endothelial cells with a soluble form of an EGF receptor (ErbB-1) or tyrphostin AG1478, a specific blocker of EGF receptor tyrosine kinase. Finally, addition of TGF-alpha alone poorly stimulated angiogenesis; however, by reducing cell death, it strongly potentiated the action of TGF-beta1. We therefore propose that TGF-beta1 promotes angiogenesis at least in part via the autocrine secretion of TGF-alpha, a cell survival growth factor, activating PI3K/Akt and p42/p44 MAPK.     

Transforming growth factor beta1 regulates follistatin mRNA expression during in vitro bovine granulosa cell differentiation

In order to test the hypothesis that transforming growth factor beta (TGF-beta) acts by FS regulation on bovine granulosa cells in in vitro differentiation, we analyzed the effect of TGF-beta1 on follistatin mRNA expression in three differentiation states of bovine granulosa cells. We showed a positive regulation of FS mRNA after TGF-beta1 (1 ng/ml) treatment of freshly isolated granulosa cells from small-medium antral follicles (2-8 mm). This effect was abolished by the addition of exogenous follistatin (100 ng/ml), suggesting that this effect could be mediated by activin. Although these cells showed a similar effect on FS mRNA expression after treatment with activin-A, a soluble form of activin receptor type IIA was unable to inactivate the TGF-beta effect. When we tested the TGF-beta effect on FS mRNA in different granulosa cell states, TGF-beta1 regulation was associated with progesterone production only in freshly isolated cells. The amount of total activin-A produced by first passage cells (dedifferentiated cells), was ten times smaller than the one measured in a conditioned medium from freshly isolated cells (mature cells). The TGF-beta1-dependent FS mRNA expression persisted in first passage cells without changes with FS addition. On the other hand, the BGC-1 granulosa cell line (immature cells) produced large amounts of activin-A regulated by TGF-beta1 and an invariable steady state of FS mRNAs. In summary, our results showed that FS mRNA expression is regulated by TGF-beta1 independently of activin effects in differentiated granulosa cells.     

Transforming growth factor beta regulates the expression and structure of extracellular matrix chondroitin/dermatan sulfate proteoglycans

Transforming growth factor beta (TGF-beta) increases up to 20-fold the expression of various forms of chondroitin/dermatan sulfate proteoglycan, the major type of sulfated proteoglycan present in the extracellular matrix and culture medium of various human, rodent, and mink cell types including kidney and lung fibroblasts, lung epithelial cells, preadipocytes, and skeletal muscle myoblasts. TGF-beta regulates the level and molecular size of these proteoglycans by acting simultaneously at two levels: it elevates the biosynthetic rate of the 45-kDa proteoglycan core protein in a cycloheximide- and actinomycin D-sensitive manner, and it induces an increase in the molecular mass of the glycosaminoglycan chains. These cellular responses correlate with occupancy of type III TGF-beta receptors by TGF-beta 1 and TGF-beta 2 and are not induced by other growth factors tested. The parameters of this effect of TGF-beta in kidney fibroblasts and myoblasts are ED50 = 5-10 pM TGF-beta 1 or TGF-beta 2, and t 1/2 = 6-8 h. These results identify the chondroitin/dermatan sulfate proteoglycans as a major component of mammalian mesenchymal and epithelial extracellular matrices whose expression and structure are regulated by TGF-beta.     

Transforming growth factor beta modulates epidermal growth factor-induced phosphoinositide metabolism and intracellular calcium levels

Transforming growth factor beta (TGF beta) alters the cellular response to epidermal growth factor (EGF) in a number of systems, but the underlying mechanisms for these alterations are largely unknown. We have examined second messenger formation in Rat-1 cells following treatment with EGF and/or TGF beta to determine whether the ability of TGF beta to potentiate some EGF-stimulated processes might be mediated by TGF beta-induced alterations in the signal transduction mechanism. Incubation of serum-deprived confluent Rat-1 cells with 10 ng/ml TGF beta resulted in a marked elevation of cellular inositol trisphosphate and inositol tetrakisphosphate levels, which were maximal at 4 h and maintained for at least 8 h. The effect of TGF beta on levels of inositol trisphosphate and inositol tetrakisphosphate was blocked by actinomycin D, suggesting that RNA synthesis was required for the TGF beta effect. While EGF stimulation induced a rapid and transient (5 min) rise in inositol phosphate levels in control cells, the EGF effect was considerably increased, both in magnitude and duration, by TGF beta treatment. Measurement of intracellular free Ca2+ with fura-2 demonstrated that TGF beta treatment markedly increased the EGF-stimulated rise in free Ca2+ and increased the duration of the response. The positive effects of TGF beta on EGF stimulation could not be explained on the basis of increased EGF binding to cells. We conclude that TGF beta treatment can both activate phosphatidylinositol turnover independently and also sensitize Rat-1 cells to stimulation by EGF.     

Transforming growth factor beta modulates phosphorylation of the epidermal growth factor receptor and proliferation of A431 cells.

Transforming growth factor beta (TGF-beta) increased the phosphorylation of the epidermal growth factor (EGF) receptor and inhibited the growth of A431 cells. Incubation with TGF-beta induced maximal EGF receptor phosphorylation to levels 1.5-fold higher than controls. Phosphorylation increased more prominently (4-5-fold) on tyrosine residues as determined by phosphoamino acid analysis and antiphosphotyrosine antibody immunoblotting. The kinase activity of EGF receptor was also elevated 2.5-fold when cells were cultured in the presence of TGF-beta. The antiproliferative effect of TGF-beta on A431 cells was accompanied by prolongation of G0-G1 phase and by morphological changes. TGF-beta augmented the growth inhibition of A431 cells which could be induced by EGF. In parallel, the specific EGF-induced increase in total phosphorylation of the EGF receptor was also augmented in the presence of TGF-beta. In cells cultured with TGF-beta, the phosphorylation of EGF receptor tyrosines induced by 20-min exposure to EGF was further increased 2-3-fold, suggesting additive effects upon receptor phosphorylation. EGF receptor activation by TGF-beta is characterized by kinetics quite distinct from that induced by EGF and therefore appears to take place through an independent mechanism. The TGF-beta-induced elevation in the phosphorylation of the EGF receptor may have a role in the augmented growth inhibition of A431 cells observed in the presence of EGF and TGF-beta.     

Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix

The angiogenic factor, basic fibroblast growth factor (FGF), either stimulates endothelial cell growth or promotes capillary differentiation depending upon the microenvironment in which it acts. Analysis of various in vitro models of spontaneous angiogenesis, in combination with time-lapse cinematography, demonstrated that capillary tube formation was greatly facilitated by promoting multicellular retraction and cell elevation above the surface of the rigid culture dish or by culturing endothelial cells on malleable extracellular matrix (ECM) substrata. These observations suggested to us that mechanical (i.e., tension-dependent) interactions between endothelial cells and ECM may serve to regulate capillary development. To test this hypothesis, FGF-stimulated endothelial cells were grown in chemically defined medium on bacteriological (nonadhesive) dishes that were precoated with different densities of fibronectin. Extensive cell spreading and growth were promoted by fibronectin coating densities that were highly adhesive (greater than 500 ng/cm2), whereas cell rounding, detachment, and loss of viability were observed on dishes coated with low fibronectin concentrations (less than 100 ng/cm2). Intermediate fibronectin coating densities (100-500 ng/cm2) promoted cell extension, but they could not completely resist cell tractional forces. Partial retraction of multicellular aggregates resulted in cell shortening, cessation of growth, and formation of branching tubular networks within 24-48 h. Multicellular retraction and subsequent tube formation also could be elicited on highly adhesive dishes by overcoming the mechanical resistance of the substratum using higher cell plating numbers. Dishes coated with varying concentrations of type IV collagen or gelatin produced similar results. These results suggest that ECM components may act locally to regulate the growth and pattern-regulating actions of soluble FGF based upon their ability to resist cell-generated mechanical loads. Thus, we propose that FGF-stimulated endothelial cells may be "switched" between growth, differentiation, and involution modes during angiogenesis by altering the adhesivity or mechanical integrity of their ECM.     

Transforming growth factor beta 1 is an epithelial-derived signal peptide that influences otic capsule formation.

Interactions between epithelial and mesenchymal tissues in the developing inner ear direct the formation of its cartilaginous capsule. Recent work indicates that many growth factors are distributed in the early embryo in vivo in a temporal-spatial pattern that correlates with sites of ongoing morphogenetic events. We report here that the localization of transforming growth factor beta 1 (TGF-beta 1) in both epithelial and mesenchymal tissues of the mouse inner ear between 10 and 16 days of embryonic development (E10-E16). In addition, utilizing a high-density culture system as an in vitro model of otic capsule chondrogenesis, we show that modulation of chondrogenesis by TGF-beta 1 in cultured mouse periotic mesenchyme mimics the in vitro effects of otic epithelium on the expression of chondrogenic potential. We provide evidence of a causal relationship of this growth factor to otic capsule formation in situ by demonstrating that the actual sequence of chondrogenic events that occur in the developing embryo is reproduced in culture by the addition of exogenous TGF-beta 1 peptide. Furthermore, in cultures of mesenchyme containing otic epithelium, we demonstrate the localization of endogenous TGF-beta 1, first within the epithelial tissue and later within both the epithelium and its surrounding periotic mesenchyme, contrasted to an absence of endogenous TGF-beta 1 in cultures of mesenchyme alone. Our results suggest that TGF-beta 1 is one of the signal molecules that mediate the effects of otic epithelium in influencing the formation of the cartilaginous otic capsule.     

Transforming growth factor beta1 induces differentiation in human papillomavirus-positive keratinocytes.

Human papillomaviruses (HPVs) are implicated in the etiology of anogenital cancers. Expression of the HPV E6 and E7 oncoproteins is believed to contribute to the carcinogenic process. Progressive loss of the ability to differentiate and resistance to the growth-inhibitory effects of endogenous signals also appear important in multistep tumorigenesis. Transforming growth factor beta1 (TGF-beta1) is a potent growth inhibitor for a variety of cultured cells. There have been conflicting reports on the ability of TGF-beta1 to inhibit the growth of HPV-positive keratinocytes in monolayer cultures. We have employed the organotypic (raft) tissue culture system, which more accurately mimics the in vivo cellular environment and architecture. We have investigated the TGF-beta1 response of HPV-positive keratinocytes derived from neoplastic cervical biopsies. Growth of these cell lines as raft tissues showed that many were altered in the ability to stratify and synthesize differentiation-specific proteins. When the organotypic tissues were treated with TGF-beta1, a more complete differentiation of the keratinocytes was induced. Treatment with 12-0-tetradecanoylphorbol-13-acetate gave similar results. TGF-beta1 treatment of HPV-positive raft epithelia led to a dose-dependent increase in E7 RNA expression in contrast to results from previous studies with monolayer cultures. Furthermore, TGF-beta1 interfered with the proliferation of HPV-positive cell lines grown in monolayer cultures. Our results suggest that loss of the ability to express markers of differentiation, a characteristic of malignancy, is a two-step process. The first step is reversible; the second is irreversible.     

Transforming growth factor beta 1 and androgen receptors in prostate neoplasia

OBJECTIVE: To investigate the interplay between transforming growth factor (TGF) beta 1, androgen receptors and stromal-epithelial interactions in benign prostatic hyperplasia (BPH), prostate intraepithelial neoplasia (PIN) and prostate carcinoma areas of prostate neoplasia. STUDY DESIGN: In this immunohistochemical study we investigated staining patterns and then determined the correlation between TGF-beta 1 expression and androgen receptor status in the epithelium and stroma of 60 paraffin-embedded tissues from radical prostatectomies. RESULTS: Staining patterns differed in the epithelium and stroma of tumor and peritumor prostatic tissue. TGF-beta 1 immunostaining (H-scores) in the epithelium and stroma increased significantly from BPH to PIN and from BPH to prostate carcinoma in the epithelium (P < .05), whereas androgen receptor (AR) immunoreactivity significantly (P < .05) increased from BPH to PIN to prostatic carcinoma in epithelium and stroma. TGF-beta 1 did not correlate with histologic grade of differentiation, whereas AR proteins were more strongly expressed in Gleason score 5 and 6 than score 7 tumors (P < .05). Nonlinear regression showed a significant correlation (P < .01) between TGF-beta 1 and AR expression only in the stromal compartment of PIN. CONCLUSION: These findings argue in favor of an interaction between TGF-beta 1 and AR in the early stages of prostate carcinogenesis and suggest that TGF-beta 1 plays a central role in stromal-epithelial interactions during the early stages of malignant transformation.     

Transforming growth factor beta has neurotrophic actions on sensory neurons in vitro and is synergistic with nerve growth factor.

Transforming growth factor beta (TGF beta) influences the growth and differentiation of a wide variety of nonneuronal cells (nnc) during embryogenesis and in response to wounding. In the present study TGF beta 1 and TGF beta 2 were examined for their neurotrophic actions on neonatal rat dorsal root ganglion (DRG) neurons with ganglionic nnc in dissociated cultures. TGF beta 1 and TGF beta 2 each increased both neuronal survival and levels of the peptide neurotransmitter substance P (SP) expressed per neuron as well as per culture. TGF beta 1 was maximally effective at a concentration of 40 pM, whereas TGF beta 2 was about 10-fold less potent. Survival effects promoted by simultaneous treatment with both factors were not additive. TGF beta 1 also changed the morphology and distribution of DRG nnc which resulted in clustering of DRG neurons on top of the nnc. Cotreatment of the cultures with two different anti-nerve growth factor (NGF) antibodies eliminated the neurotrophic effects of TGF beta 1. However, treatment with TGF beta 1 did not alter NGF mRNA expression in the cultures nor did it change the amount of NGF in the medium. Further, TGF beta 1 greatly enhanced survival effects and SP stimulation promoted by exogenous NGF at concentrations up to 100 ng/ml. The neurotrophic effects of TGF beta 1 were significantly attenuated by decreasing the proportion of the ganglionic nnc, suggesting a role for these cells in mediating TGF beta 1 action on the neurons. It is hypothesized that the neurotrophic activity of TGF beta depended upon the presence of molecules immunologically related to NGF and that the effects of TGF beta were synergistic with NGF. These observations suggest that TGF beta may play a role in the differentiation and regeneration of DRG neurons in vivo.     

Regulation of extracellular matrix proteins by transforming growth factor beta1 in cultured pulmonary endothelial cells

Transforming growth factor beta-1 (TGF-beta1), which is present in lung tissue, has been suggested to play a role in modulating vascular cell function in vivo. The action of TGF-beta1 in vivo, especially at the local site of application to connective tissue, is anabolic and leads to pulmonary fibrosis and angiogenesis, strongly indicating that TGF-beta may have practical applications in repair of tissue injury caused by burns, trauma, or surgery. In the present study, we have used cultured bovine pulmonary artery endothelial (BPAE) cells as a model system. Expression of various proteins, including SPARC (secreted protein acidic and rich in cysteines), type IV procollagen and fibronectin (FN) was examined by radiolabeling the cells with [3H]proline, immunoprecipitation with specific antibodies, and Northern blot analyses by using specific cDNA probes. Cultured cells were labeled with [3H]proline for 24 h in either the absence or in the presence of TGF-beta1 (0-20 ng/ml). Incorporation of radioactivity was observed in a concentration-dependent manner, maximal at 5 ng/ml. Northern blot hybridization demonstrated that TGF-beta1 (5 ng/ml) treatment of BPAE cells caused an increase in steady-state levels     

Spatial organization of extracellular matrix and fibroblast activity: effects of serum, transforming growth factor beta, and fibronectin

The goal of our research is to understand reciprocal relationships between cell function and tissue organization. We studied the regulation of fibroblast activity in an in vitro culture model that recapitulates in continuous fashion the cycle of events occurring during connective tissue repair. We present evidence that concomitant with spatial reorganization of the extracellular matrix, there was a dramatic decline in extracellular matrix synthesis and cell proliferation. Therefore, spatial reorganization was a crucial turning point for fibroblast activity. Factors that regulated the timing of spatial reorganization included serum, transforming growth factor beta, and fibronectin. By accelerating spatial reorganization of the cultures, transforming growth factor beta led to a relative decrease in cell proliferation and extracellular matrix synthesis. By retarding spatial reorganization of the cultures, fibronectin led to a relative increase in cell proliferation and extracellular matrix synthesis. The results indicate that spatial information in the three-dimensional cell-matrix interaction permits higher order, tissue-level regulation of fibroblast function.     

Transforming growth factor beta 1 inhibits epidermal growth factor receptor endocytosis and down-regulation in cultured fetal rat hepatocytes.

Incubation of fetal rat hepatocytes (FRH) with transforming growth factor beta 1 (TGF-beta 1) resulted in growth arrest and a biphasic effect on epidermal growth factor (EGF) receptor. After 2 h of exposure, EGF receptor (EGFR) was reduced by 43%. From 6 to 24 h, TGF-beta 1 exposure resulted in progressive increase in EGFR up to 74% over control. The increased binding was due to increase in high affinity EGF binding sites. FRH grown in medium containing EGF exhibited down-regulated EGFR with loss of high affinity EGF binding sites. With TGF-beta 1 exposure, high affinity EGFR was not down-regulated by EGF. Since down-regulation of EGFR involves internalization, the kinetics of EGF receptor-mediated endocytosis were examined. In TGF-beta 1-exposed FRH, EGF endocytosis was inhibited, with a reduction in the first order rate constant for the process from 0.078 to 0.043 min-1. Despite inhibition of growth, receptor down-regulation, and EGF endocytosis after TGF-beta 1 exposure, EGF-induced receptor autophosphorylation was preserved as demonstrated by [32P]phosphate-labeling of immunoprecipitated EGFR. These observations provide direct evidence that TGF-beta 1 regulates growth of fetal cells. Further, they suggest that TGF-beta 1 regulates endocytosis of EGF and possibly of other ligands.     

Transforming growth factor beta 1-induced changes in cell migration, proliferation, and angiogenesis in the chicken chorioallantoic membrane

Application of TGF beta 1 (10-100 ng) to the chicken chorioallantoic membrane (CAM) for 72 h resulted in a dose-dependent, gross angiogenic response. The vascular effects induced by TGF beta 1 were qualitatively different than those induced by maximal doses of basic FGF (bFGF) (500 ng). While TGF beta 1 induced the formation of large blood vessels by 72 h, bFGF induced primarily small blood vessels. Histologic analysis revealed that TGF beta 1 stimulated pleiotropic cellular responses in the CAM. Increases in fibroblast and epithelial cell density in the area of TGF beta 1 delivery were observed as early as 4 h after TGF beta 1 treatment. By 8 h, these cell types also demonstrated altered morphology and marked inhibition of proliferation as evidenced by 3H-thymidine labeling. Thus, the TGF beta 1-stimulated accumulation of these cell types was the result of cellular chemotaxis from peripheral areas into the area of TGF beta 1 delivery. Microscopic angiogenesis in the form of capillary sprouts and increased endothelial cell density first became evident at 16 h. By 24 h, capillary cords appeared within the mesenchyme of the CAM, extending towards the point of TGF beta 1 delivery. 3H-thymidine labeling revealed that the growth of these capillary cords was due to endothelial cell proliferation. Finally, perivascular mononuclear inflammation did not become evident until 48 h of treatment, and its presence correlated spatially and temporally with the gross and histological remodelling of newly formed capillary cords into larger blood vessels. In summary, these data suggest that, in the chicken CAM, TGF beta 1 initiates a sequence of cellular responses that results in growth inhibition, cellular accumulation through migration, and microvascular angiogenesis.     

Transforming growth factor beta in cardiovascular development and function

Transforming growth factor betas (TGFbetas) are pleiotropic cytokines involved in many biological processes. Genetic engineering and tissue explanation studies have revealed specific non-overlapping roles for TGFbeta ligands and their signaling molecules in development and in normal function of the cardiovascular system in the adult. In the embryo, TGFbetas appear to be involved in epithelial-mesenchymal transformations (EMT) during endocardial cushion formation, and in epicardial epithelial-mesenchymal transformations essential for coronary vasculature, ventricular myocardial development and compaction. In the adult, TGFbetas are involved in cardiac hypertrophy, vascular remodeling and regulation of the renal renin-angiotensin system. The evidence for TGFbeta activities during cardiovascular development and physiologic function will be given and areas which need further investigation will be discussed.