Rat Heart Endothelial Cells Research Articles

Upregulation of vascular endothelial growth factor by H 2O 2 in rat heart endothelial cells

Hydrogen peroxide (H 2O 2) is a reactive oxygen species generated by several metabolic pathways in mammalian cells. Endothelial cells are extremely susceptible to oxidative stress. H 2O 2 has been reported to increase the permeability in these cells. Using rat heart endothelial cell culture as a model system, we examined the effect of H 2O 2 on the gene expression of vascular endothelial growth factor (VEGF), a potent mitogen of endothelial cells and a vascular permeability factor. By Northern blot analysis we found that VEGF mRNA responded to H 2O 2 in a dose-and time-dependent manner. The induction was superinduced by cycloheximide and blocked by actinomycin D. N-Acetylcysteine, a synthetic antioxidant, was able to suppress the induction. H7, a protein kinase C inhibitor, could also block the induction. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors, AP-1 and NF-κB. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 4 h after H 2O 2 stimulation. Our results demonstrate that VEGF gene expression is upregulated by H 2O 2 in these endothelial cells.

Association of malondialdehyde–acetaldehyde (MAA) adducted proteins with atherosclerotic-induced vascular inflammatory injury

Atheroscierosis is a vascular injury characterized by elevated tissue levels of tumor necrosis factor- α (TNF- α), increased expression of endothelial cell adhesion molecules, and vascular wall inflammatory cell infiltration. Foam cells are associated with atherosclerotic plaque material, and low density lipoprotein (LDL) is a lipid component of foam cells. Malondialdehyde (MDA) is an oxidative product of unsaturated fatty acids and is also present in atherosclerotic lesions. MDA-modified (adducted) proteins, including MDA-modified LDL, are present in atherosclerotic human vascular tissue. Acetaldehyde (AA) is the major metabolic product of ethanol oxidation. Both MDA and AA are highly reactive aldehydes and will combine with proteins to produce an antigenically distinct protein adduct, termed the MAA adduct. This study demonstrates that proteins modified in the presence of high concentrations of MDA can produce MAA-modified proteins in vitro. In addition, MAA adducted proteins are capable of inducing rat heart endothelial cell cultures (rHEC) to produce and release TNF- α, and cause rHEC upregulation of endothelial adhesion molecule expression, including ICAM-1. These adhesion molecules are required for circulating inflammatory cells to adhere to endothelium which allows inflammatory cell tissue infiltration. Additionally, MAA modified proteins were defected in human atherosclerotic aortic vascular tissue but not in normal aortic tissue. Since atherosclerosis is associated with an inflammatory vascular injury characterized by elevated tissue TNF- α concentrations and inflammatory cell infiltration, these data suggest that MAA-adducted proteins may be formed in atherosclerotic plaque material and may be involved in the inflammatory reaction that occurs in atherosclerosis. These data further suggest that previous studies demonstrating MDA modified protein in atherosclerotic plaque may in fact have MAA modified proteins associated with them.

2-Phenyl-4-quinolone prevents serotonin-induced increases in endothelial permeability to albumin

To investigate the role of 2-phenyl-4-quinolone in enhancing endothelial monolayer paracellular barrier function and preventing the disturbance of paracellular barrier function by vasoactive agents, the study examined the effect of 2-phenyl-4-quinolone on serotonin-mediated macromolecule transfer and microfilament changes in cultured rat heart endothelial cells. Serotonin-treated endothelial cells induced concentration-dependent increases in the passage of Evans blue dye-bound bovine serum albumin. Incubation of the endothelial monolayers with 2-phenyl-4-quinolone antagonized serotonin- and cytochalasin B-induced macromolecular permeability. 2-Phenyl-4-quinolone also opposed the effect of serotonin or cytochalasin B on the distribution and quantity of actin filaments in the endothelial cytoskeleton. Furthermore, 2-phenyl-4-quinolone alone led to an apparent quantitative increase in F actin fluorescence in endothelial cells. The addition of 10 −7 M 2-phenyl-4-quinolone had an effect on serotonin-induced changes in the myosin and distribution of myosin were comparable to that on serotonin monolayers. In conclusion, 2-phenyl-4-quinolone attenuated the serotonin-induced permeability of rat heart endothelial cells and this was associated with stabilization of F actin microfilaments and changes in the myosin organization. This result suggests that influences on cytoskeletal assembly may be involved in this process.

Decreased protein kinase C activation mediates inhibitory effect of norathyriol on serotonin-mediated endothelial permeability

We examined the mechanisms of norathyriol on the serotonin-induced increased permeability of rat heart endothelial cell monolayers. The present study showed that the activation of rat heart endothelial cell protein kinase C by phorbol myristate acetate led to the dose-dependent increase in endothelial permeability to albumin, an effect that was inhibited by staurosporine (a protein kinase inhibitor). Staurosporine also attenuated the serotonin-induced increase in permeability. Norathyriol abolished both serotonin- and phorbol myristate acetate-induced permeability. We investigated whether norathyriol, by inhibiting protein kinase C activation, attenuated the serotonin-induced permeability. Immunofluorescence studies demonstrated that norathyriol prevented the redistribution of protein kinase C isozymes following stimulation with serotonin. Western blot analysis showed that norathyriol significantly inhibited the serotonin-induced translocation of the α protein kinase C isozyme from the cytosolic to the particulate fraction. In conclusion, norathyriol attenuates the serotonin-induced permeability of rat heart endothelial cells to macromolecules in association with inhibition of protein kinase C activation. This decrease in endothelial cell permeability may be one of the mechanisms for the protective effects of norathyriol against edema formation in response to inflammatory agonists in vivo.

Norathyriol Prevents Serotonin-induced Increases in Endothelial Permeability to Albumin

To investigate the role of norathyriol in enhancing the endothelial monolayer paracellular barrier function, and preventing disturbance of the paracellular barrier function by vasoactive agents, this study examined the effect of norathyriol on serotonin-mediated macromolecule transfer and microfilament change in cultured rat heart endothelial cells (RHEC). Incubation of the endothelial monolayer with norathyriol antagonized serotonin-and cytochalasin B-induced macromolecular permeability. Norathyriol also opposed the effect of serotonin on the distribution of actin filaments in the endothelial cytoskeleton. Upon addition of 10^(-6) M norathyriol, serotonininduced changes in the myosin and distribution of myosin were comparable to that of serotonin monolayers. In conclusion, norathyriol decreased the permeability of RHEC caused by serotonin, which was associated with the stabilization of F-actin microfilaments and changes in the myosin organization, suggesting that influences on cytoskeletal assembly may be involved in this process.

Upregulation of vascular endothelial growth factor by angiotensin II in rat heart endothelial cells

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and a vascular permeability factor. In this study we found that the addition of angiotensin II (AII) to rat heart endothelial cells induced VEGF mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of AII and decreased after 4 h. The induction was superinduced by cycloheximide and blocked by actinomycin D. Losartan, an AT 1 receptor antagonist, abolished the induction of VEGF mRNA by AII, whereas PD 123319, an AT 2 receptor antagonist, had no effect on VEGF mRNA induction. H7, a protein kinase C inhibitor, blocked the induction. RT-PCR experiments showed two mRNA species (VEGF 120 and VEGF 164) in these cells and both species were stimulated by AII. Transient transfection experiment showed that VEGF promoter activity was increased 2.2-fold upon AII stimulation. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-κB. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 8 h after AII stimulation. Our results demonstrate for the first time that the upregulation of VEGF by AII may play a significant role in AII-induced hyperpermeability.

Synthesis, storage and regulated secretion of tissue-type plasminogen activator by cultured rat heart endothelial cells

The synthesis, storage and regulated secretion (acute release) of tissue-type plasminogen activator (tPA) were studied in cultured rat heart endothelial (RHE) cells. In this study, special attention was paid to the correspondence between tPA metabolism in RHE cells in vitro and tPA metabolism in rats in vivo. RHE cells synthetized tPA, as shown by a spectrophotometric activity assay, by fibrin autography and by an ELISA for tPA antigen. The synthesis of tPA was strongly enhanced by phorbol myristate acetate, by all-trans-retinoic acid, by 8-bromoadenosine 3′:5′ cyclic-monophosphate (8-br-cAMP) and by compounds that enhance cAMP synthesis, such as cholera toxin and forskolin. Urokinase-type plasminogen activator and plasminogen activator inhibitor were not detected. tPA was constitutively secreted by RHE cells, but was also present in an intracellular pool of small dense granules, from which it could be acutely released by stimulating the cells with thrombin or the calcium ionophore A-23187. This release was maximal during the first minutes after stimulation. In all these aspects of tPA metabolism, RHE cells closely resembled rat endothelial cells in vivo. It is concluded that cultured RHE rat heart endothelial cells constitute a relevant in vitro model for studying endothelial tPA metabolism.

Mast cell-mediated induction of ICAM-1, VCAM-1 and E-selectin in endothelial cells in vitro: constitutive release of inducing mediators but no effect of degranulation.

Mast cell (MC)-mediated induction of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and of E-selectin was studied in cultures of rat heart endothelial cells (EC) and human umbilical vein EC (HUVEC) respectively. MC induced VCAM-1 and E-selectin, but hardly any ICAM-1 in EC. Induction was not dependent on MC degranulation, but seemed to be provoked by constitutively released substances, other than histamine, from MC. Co-incubation of MC and EC, allowing for direct contact between the two cell types, was more potent in induction than MC co-incubated separately from EC using a permeable membrane. MC were less potent in induction than exogenous added cytokines or LPS. Induction of cell adhesion molecules in rat heart EC was MC-specific, since EC incubations with either rat cardiomyocytes or heart fibroblasts had no effect. The data show that rat MC, independent of degranulation, secrete mediators relevant for the induction of a specific set of EC adhesion molecules in vitro. This suggests a (supportive) role for MC in cell-adhesion molecule induction in the endothelium in settings of early or mild inflammation. The results are discussed in the context of inflammatory processes in the heart in vivo.

Inhibitory effect of 2-phenyl-4-quinolone on serotonin-mediated changes in the morphology and permeability of endothelial monolayers

The integrity of endothelial cell monolayers, a critical requirement for barrier maintenance, is needed for the prevention of edema formation. To investigate the mechanisms by which 2-phenyl-4-quinolone (YT-1) provided protection against serotonin-induced exudation, rat heart endothelial cell cultures were used. In this study, serotonin and phorbol myristate acetate (PMA) caused endothelial cells to became permeable to macromolecules by causing cell contraction and intercellular gap formation. These responses were attenuated by staurosporine, a protein kinase C inhibitor. Further experiments showed that YT-1 (1) did not alter serotonin-mediated early signal events such as protein kinase C activation, (2) protected against serotonin-induced endothelial barrier dysfunction by increasing intracellular cAMP levels, (3) played a role in regulating adenylate cyclase activity, (4) reversed serotonin-induced permeability to macromolecules, an effect which did not correlate with intracellular cGMP concentrations. This study demonstrates a possible mechanism by which YT-1 protects endothelial function and preserves the microvasculature from pharmacologic injury by vasoactive agents.

Regulation of thrombospondin-1 production by angiotensin II in rat heart endothelial cells

Thrombospondin-1 (TSP-1) is synthesized, secreted, and incorporated into the extracellular matrix by a variety of cells, including the endothelial cells. Addition of angiotensin II (AII) significantly induced TSP-1 mRNA in rat heart-derived endothelial cells. TSP-1 mRNA levels reached a plateau within 2 h after the addition of AII and decreased after 5 h. The induction was superinduced by cycloheximide and blocked by actinomycin D. Losartan, an AT1 receptor antagonist, could abolish the induction of TSP-1 mRNA by AII. Phorbol 12-myristate 13-acetate (TPA) was found to enhance TSP-1 mRNA level whereas a protein kinase C inhibitor, H7, was shown to block the induction. Immunoblot analysis revealed that TSP-1 was detectable in the medium 4 h after AII stimulation. Our results suggest that the upregulation of TSP-1 by AII represents an important mechanism leading to perivascular fibrosis in the heart.

Intracellular thiol redox status affects rat cytomegalovirus infection of vascular cells

There is increasing evidence for cytomegalovirus (CMV) induced vascular pathology during acute infection in the immunocompromised host. Inflammation is involved in such processes, which is frequently associated with increased levels of oxidative mediators and reduced anti-oxidant protection. A relation between viral infection and oxidative stress has been recognized for human immunodeficiency virus and herpes simplex virus-1 infections, but little is known in this respect for CMV infections. We investigated if there is a relation between CMV infection of vascular cells and the intracellular redox status using an in vitro rat model. We measured intracellular glutathione levels and rat CMV (RCMV) permissiveness of rat heart endothelial cell lines (RHEC), rat smooth muscle cells (RSMC), and compared these with fully CMV-permissive rat fibroblasts (REF and Rat 2). In addition, the effects of the anti-oxidant N-acetylcysteine (NAC) and the glutathione synthesis inhibitor buthionine sulfoximide (BSO) on CMV permissiveness and replication were investigated in these cell lines. Finally, we investigated infection of vascular cells under inflammatory conditions in an in vivo rat model for acute CMV infection. The results show a very high endogenous glutathione level in RHEC compared to REF, Rat 2 cells and RSMC. This is associated with a low CMV permissiveness in RHEC as opposed to full permissiveness in REF, Rat 2 cells and RSMC in vitro. In addition, modulation of the intracellular thiol redox status affected CMV infection and replication only in RHEC, but not in RSMC and Rat 2 cells. During acute infection in vivo under immunosuppressed conditions rat endothelial cells first become activated and subsequently infected leading to vascular damage and pathology. This study suggests that a high endogenous thiol redox status may contribute to the apparent barrier function of endothelial cells with respect to CMV infection and that oxidative stress may facilitate CMV infection of the vascular wall.

Angiotensin II induces TIMP-1 production in rat heart endothelial cells.

Angiotensin II (AII) was found to upregulate tissue inhibitor of metalloproteineses-1 (TIMP-1) gene expression in rat heart endothelial cells in a dose and time-dependent manner. The maximal stimulation of TIMP-1 mRNA was achieved by 2 h after the addition of AII. This effect was blocked by losartan, an AT1 receptor antagonist and by calphostin C, a protein kinase C inhibitor. Addition of cycloheximide superinduced and actinomycin D abolished the induction. These results suggest that AII stimulates TIMP-1 production by a protein kinase C dependent pathway which is dependent upon de novo RNA synthesis. Immunoprecipitation experiment showed an enhanced band of 28 kDa from the conditioned medium of AII-treated cultures. Immunoblot analysis revealed that TIMP-1 was detectable in the conditioned medium 4 h after AII stimulation. Since endothelial cells line the blood vessels and sense the rise in AII associated with hypertension, the TIMP-1 released by these cells may provide an initial trigger leading to cardiac fibrosis in angiotensin-renin dependent hypertension.

Angiopeptin, a somatostatin-14 analogue, decreases adhesiveness of rat Leukocytes to unstimulated and IL-1β-activated rat heart endothelial cells

We have previously demonstrated that somatostatin-14 and its octapeptide analogue, angiopeptin, decrease the ability of rat heart endothelial cells to bind leukocytes [Leszczynski, et al., Reg. Pept. 43 (1993) 131–140]. Here, we examined whether exposure of leukocytes to angiopeptin modifies their adhesiveness to the unstimulated and to IL-1β-activated endothelium. Monolayers of unstimulated endothelial cells bind 274 ± 12 leukocytes/mm 2. Exposure of leukocytes for 1, 4 and 24 hours to angiopeptin (1 μM) reduced significantly (p < 0.05) adhesion of leukocytes from 274 ± 12 to 188 ± 10, 185 ± 8 and 172 ± 3 cells/mm 2, respectively. Stimulation of endothelial cells with IL-1β (100U/ml) for 24 hours increased endothelial adhesiveness from 274 +- 12 to 381 ± 17 adhering leukocytes/mm 2. Exposure of leukocytes for 1, 4 and 24 hours to angiopeptin (1μM) reduced significantly (p < 0.05) binding of leukocytes to IL-1β-activated endothelium from 381 ± 17 to 237 ± 8, 254 ± 11 and 248 ± 13 cells/mm 2, respectively. Angiopeptin had no effect on the expression of lymphocyte function-associated molecule-1 (LFA-1; CDlla/CD18) by leukocytes, as assessed by flow cytometry. This suggests that angiopeptin modulates adhesive properties of leukocytes by (1) altering the expression of other than LFA-1 adhesion molecule(s) and/or (2) modulating the affinity of adhesion molecule(s) expressed by leukocytes. In conclusion, our results demonstrate that angiopeptin reduces leukocyte adhesiveness to unstimulated and to IL-1β-activated endothelium. It suggests that angiopeptin may suppress immune response via modulation of the leukocyte-endothelial interaction.

Stimulation of a Ca2+-Calmodulin-activated Histone 3 Arginine Kinase in Quiescent Rat Heart Endothelial Cells Compared to Actively Dividing Cells

A Ca(2+)-calmodulin-activated histone 3 kinase was partially purified from nuclear extracts of dividing and quiescent rat heart endothelial cells. The histone 3 phosphorylating activity was 20-100-fold higher in quiescent than in dividing cells. Base hydrolysis followed by amino acid analysis revealed that histone 3 was phosphorylated on arginine. Further investigations were conducted to determine whether phosphorylation of histone 3 also occurred in vivo. Cells were incubated for 3 h in a phosphate-free medium supplemented with [32P]phosphoric acid. It was observed that the nuclear content of arginine-phosphorylated histone 3 was considerably higher in quiescent than in dividing rat heart endothelial cells. The histone 3 arginine kinase is a component of a complex containing a Ca(2+)-dependent calmodulin-binding protein of apparent molecular mass of 85 kDa. Using polyclonal antibodies to an 85-kDa protein, also the major Ca(2+)-dependent calmodulin-binding component of the histone 3 arginine kinase from calf thymus, an immunoreactive protein of identical apparent molecular mass was found to be present in equal amounts both in dividing and quiescent cells. We propose that the 85-kDa protein is either the histone 3 arginine kinase or one of its subunits and that phosphorylation of histone 3 is involved with cell cycle exit in eukaryotes.

Isolation of rat heart endothelial cells and pericytes: Evaluation of their role in the formation of extracellular matrix components

In order to facilitate investigation of the cells responsible for overproduction of type VI collagen in the extracellular matrix surrounding the capillaries of diabetic rat myocardium, procedures have been developed for the isolation from this tissue of endothelial cells as well as a cell type identified as pericytes. This was accomplished by enzymatic and mechanical disruption of ventricles from young rats (125 g) followed by removal of myocytes through their nonadherence to tissue culture surfaces. Endothelial cells were separated by fluorescence-activated cell sorting after staining with rhodamine-labeled acetylated low density lipoprotein and were identified by their monolayer growth pattern, reaction with anti-von Willebrand factor and the ability to form capillary-like tubes induced by low serum concentration. Pericytes were purified by selective scraping for removal of other cell types and were identified by their irregular shape, overlapping growth pattern at confluence, reaction with anti-smooth muscle actin and content of GLUT4 glucose transporter. Fibroblasts, visualized after staining with rhodamine-labeled α2-macroglobulin, were only rarely detected. Analysis of collagen by immunoblotting indicated formation by both cell types of α1(IV) collagen as well as the three subunits of type VI (α3 at 205 kDa and α1 plus α2 at 150 kDa). Both endothelial cells and pericytes demonstrated transcripts for types VI, IV and I collagen, as well as fibronectin, but while the level of the mRNA for type IV collagen was higher in pericytes than in endothelial cells, the reverse was true for collagens VI and I and fibronectin. These observations suggest that both endothelial cells and pericytes contribute to formation of the myocardial capillary matrix, but that changes involving only type VI collagen, such as occur in diabetic cardiomyopathy, may reflect a response primarily of endothelial cells.

Effect of high glucose on formation of extracellular matrix components by cultured rat heart endothelial cells

In an attempt to define the basis for the microvascular changes observed in diabetic myocardium, a study was undertaken on the effect of elevated glucose on the synthesis by rat heart endothelial cells of the extracellular matrix components, types VI, IV and I collagen, as well as fibronectin. Confluent cultures of these cells, isolated by fluorescence-activated cell sorting after treatment with rhodamine-labelled acetylated low density lipoprotein, showed a three to fivefold enhancement in the synthesis of type VI collagen after exposure for 48 h to high glucose (20 to 30 mmol/l), as determined by immunoblot analysis. Increased production of type IV collagen and fibronectin was also observed, but the change was smaller and no effect on type I collagen was found. Measurement of mRNA levels by hybridization with cDNA probes indicated that 48-h exposure to high glucose significantly increased the level of transcripts for type VI and IV collagens but not for type I collagen. While glucose consumption by endothelial cells in high glucose doubled in the initial 24-h period, utilization returned to normal by 48 h, concomitant with a reduction in GLUT1 transcript levels, suggesting that signals for stimulation of collagen synthesis must be active during the initial period of exposure to elevated glucose levels.

Effect of high glucose on formation of extracellular matrix components by cultured rat heart endothelial cells

Effect of high glucose on formation of extracellular matrix components by cultured rat heart endothelial cells

Fatty Acid Transfer Across the Myocardial Capillary Wall: No Evidence of a Substantial Role for Cytoplasmic Fatty Acid-binding Protein

It has recently been hypothesized that fatty acid (FA) transfer across the myocardial capillary wall is mediated by cytoplasmic fatty acid-binding protein (FABP). Therefore, we studied the type and content of FABP in endothelial cells from rat heart, using molecular biological, immunochemical, and FA-binding assays. Studies were performed on short term cultured endothelial cells, two established endothelial cell lines and ultrathin cryosections from adult rat heart. Northern blotting analysis of endothelial cell RNA failed to detect either heart-type (H-) FABP or liver-type (L-) FABP mRNA, but the reversed transcription-polymerase chain reaction revealed both H- and L- FABP mRNAs, indicating the presence of minor amounts of these mRNAs. Highly sensitive immunochemical assays (sandwich ELISAs) using specific antibodies raised against rat H- or L-FABP showed the contents of these FABP-types in endothelial cells to be 1-5 ng/mg cytosolic protein, which is more than three orders of magnitude lower than the contents of H-FABP in heart or L-FABP in liver. Immuno-electron microscopy also showed that the concentration of H-FABP in endothelial cells is at least two orders of magnitude lower than that in cardiomyocytes. Finally, cytosolic protein samples from endothelial cells revealed no significant FA-binding activity in the 15-kDa region. We conclude that rat heart endothelial cells contain only minor quantities of cytoplasmic FABP and that, therefore, FA transport over the endothelium is mediated by FABP only to a minor extent. It is postulated that aqueous diffusion of FA through the endothelial cytoplasm most likely accounts for the experimentally observed rates of cardiac FA utilization.

Induction of class II molecules by cytomegalovirus in rat heart endothelial cells is inhibited by ganciclovir.

Cytomegalovirus (CMV) has been demonstrated to induce class II antigen expression in endothelial cells. To study whether ganciclovir (DHPG) has an effect on CMV-induced class II expression, cultured rat heart endothelial cells were infected with rat CMV (RCMV) and treated with different DHPG concentrations. Class II antigens in endothelial cells were detected by a monoclonal antibody and immunoperoxidase technique. Control cells did not express class II antigen, but during RCMV infection 92% of cells were class II-positive. DHPG treatment (1, 10, 100 and 1000 microg/ml) decreased RCMV-induced class II expression from 73% to 59%, 6% and 0%, respectively. As DHPG inhibits CMV DNA polymerase, our present results suggest that DHPG affects RCMV-induced class II expression via the inhibition of RCMV DNA replication.

Angiotensin II induces TGF-β1 production in rat heart endothelial cells

Angiotensin II (AII) was found to stimulate TGF-β1 gene expression in rat heart endothelial cells in a dose- and time-dependent manner. The maximal induction of TGF-β1 mRNA was achieved by 6 h after the addition of AII. This induction was blocked by losartan, an AT1 receptor antagonist and by calphostin C, a protein kinase C inhibitor. Addition of actinomycin D and cycloheximide abolished the induction. TGF-β1 promoter activities were stimulated 5-fold by AII. TGF-β1 secreted by the rat heart endothelial cells in response to AII was in a latent form and could be activated by mild heat treatment. These results suggest that AII stimulates TGF-β1 production by a protein kinase C-dependent pathway which is dependent upon de novo RNA synthesis and protein synthesis. Since endothelial cells line the blood vessels and sense the rise in AII associated with hypertension, the release of TGF-β1 by these cells may provide the initial trigger leading to cardiac fibrosis in angiotensin-renin-dependent hypertension.