The molecular basis of increased glomerulosclerosis after blockade of the renin angiotensin system in growth hormone transgenic mice.

BACKGROUND: Angiotensin converting enzyme inhibitor (ACEi) therapy delays the onset of renal failure in diabetic nephropathy and inhibits or delays the onset of proteinuria in several animal models. MATERIALS AND METHODS: We examined this question using a transgenic model of chronic glomerulosclerosis caused by an excess production of growth hormone (GH) in which there is progressive glomerular scarring leading to uremia. In addition, since GH mice do not have systemic hypertension or an elevated glomerular filtration rate, we could address the question of whether ACEi or angiotensin II receptor antagonists (AII RA) had an effect on the development of glomerulosclerosis under these conditions. Since excess matrix accumulates in glomerulosclerosis because of alterations in the balance between its synthesis and degradation, we examined the effect of ACEi and AII RA on these parameters. RESULTS: Systemic blood pressure was unaffected by ACEi treatment, but the glomerular filtration rate decreased 85%. ACEi-treated mice had increased mesangial deposition of type I collagen and decreased 105 kD complex collagenase activity. In addition, ACEi-treated GH mice had increased glomerular alpha 1 type I collagen, alpha 1 type IV collagen, and alpha-smooth muscle cell actin mRNAs. No changes were noted in beta actin, or 72 kD metalloproteinase mRNAs. The result of these changes was a net increase in sclerosis. Surprisingly, GH mice treated with ACEi or AngII RA developed marked renal arteriolar lesions. CONCLUSIONS: In some forms of glomerulosclerosis, the lesions develop independently of angiotensin II. Pharmacological inhibition of angiotensin II, in this circumstance, may aggravate the lesions through disregulation of the levels and the balance between glomerular matrix synthesis and degradation.     

Temporal changes in renal endoglin and TGF-β1 expression following ureteral obstruction in rats

Chronic renal disease is characterized by the accumulation of extracellular matrix proteins in the kidney and a loss of renal function. Tubulointerstitial fibrosis has been reported to play an important role in the progression of chronic renal diseases. Transforming growth factor-beta1 (TGF-beta1) is a profibrotic cytokine playing a major contribution to fibrotic kidney disease. Endoglin is a membrane glycoprotein of the TGF-beta1 receptor system. The aim of this work was to determine the time-course expression of renal type I and IV collagens, endoglin and TGF-beta1 in a rat model of induced tubulointerstitial fibrosis at 1, 3, 10 and 17 days after unilateral ureteral obstruction (UUO). In 17 days-ligated (L)-renal samples, a marked interstitial fibrosis was detected by Masson's trichromic and Sirius red staining, accompanied by an increase in type I collagen expression as shown by immunohistochemical analysis. Northern blot studies revealed a progressive increase in collagen alpha2(I), TGF-beta1 and endoglin mRNA expression in L kidneys when compared with the corresponding non-ligated (NL) kidneys from the animals subjected to left UUO. Seventeen days after UUO, significant increases in collagen alpha2(I), collagen alpha1(IV), TGF-beta1 and endoglin mRNA levels were detected in L kidneys vs NL kidneys. Significantly higher levels of the protein endoglin were found in L kidneys than in NL kidneys 10 and 17 days following obstruction. A marked increase expression for endoglin and TGF-beta1 was localized in renal interstitium by immunohistochemical studies 17 days after obstruction. In conclusion, this work reports the upregulation of endoglin coincident to that of its ligand TGF-beta1 in the kidneys of rats with progressive tubulointerstitial fibrosis induced by UUO.     

Iron chelation and a free radical scavenger suppress angiotensin II-induced upregulation of TGF-beta1 in the heart

Long-term administration of angiotensin II causes myocardial loss and cardiac fibrosis. We previously found iron deposition in the heart of the angiotensin II-infused rat, which may promote angiotensin II-induced cardiac damage. In the present study, we have investigated whether an iron chelator (deferoxamine) and a free radical scavenger (T-0970) affect the angiotensin II-induced upregulation of transforming growth factor-beta1 (TGF-beta1). Angiotensin II infusion for 7 days caused a robust increase in TGF-beta1 mRNA expression in vascular smooth muscle cells, myofibroblast-like cells, and migrated monocytes/macrophages. T-0970 and deferoxamine suppressed the upregulation of TGF-beta1 mRNA and reduced the extent of cardiac fibrosis in the heart of rats treated with angiotensin II. These agents blocked the angiotensin II-induced upregulation of heme oxygenase-1, a potent oxidative and cellular stress-responsive gene, but they did not significantly affect systolic blood pressure or plasma levels of aldosterone. In addition, T-0970 and deferoxamine suppressed the angiotensin II-induced upregulation of monocyte chemoattractant protein-1 in the heart. These results collectively suggest that iron and the iron-mediated generation of reactive oxygen species may contribute to angiotensin II-induced upregulation of profibrotic and proinflammatory genes, such as TGF-beta1 and monocyte chemoattractant protein-1.     

Transforming growth factor-beta1 stimulates collagen matrix remodeling through increased adhesive and contractive potential by human renal fibroblasts

Renal tubulointerstitial fibrosis is the common final pathway leading to end-stage renal failure. Tubulointerstitial fibrosis is characterized by fibroblast proliferation and excessive matrix accumulation. Transforming growth factor-beta1 (TGF-beta1) has been implicated in the development of renal fibrosis accompanied by alpha-smooth muscle actin (alpha-SMA) expression in renal fibroblasts. To investigate the molecular and cellular mechanisms involved in tubulointerstitial fibrosis, we examined the effect of TGF-beta1 on collagen type I (collagen) gel contraction, an in vitro model of scar collagen remodeling. TGF-beta1 enhanced collagen gel contraction by human renal fibroblasts in a dose- and time-dependent manner. Function-blocking anti-alpha1 or anti-alpha2 integrin subunit antibodies significantly suppressed TGF-beta1-stimulated collagen gel contraction. Scanning electron microscopy showed that TGF-beta1 enhanced the formation of the collagen fibrils by cell attachment to collagen via alpha1beta1 and alpha2beta1 integrins. Flow cytometry and cell adhesion analyses revealed that the stimulation of renal fibroblasts with TGF-beta1 enhanced cell adhesion to collagen via the increased expression of alpha1 and alpha2 integrin subunits within collagen gels. Fibroblast migration to collagen was not up-regulated by TGF-beta1. Furthermore, TGF-beta1 increased the expression of a putative contractile protein, alpha-SMA, by human renal fibroblasts in collagen gels. These results suggest that TGF-beta1 stimulates fibroblast-collagen matrix remodeling by increasing both integrin-mediated cell attachment to collagen and alpha-SMA expression, thereby contributing to pathological tubulointerstitial collagen matrix reorganization in renal fibrosis.     

The involvement of transforming growth factor-beta1 secretion in urotensin II-induced collagen synthesis in neonatal cardiac fibroblasts

As the most potent vasoconstrictor in mammals, urotensin II (U II) has recently been demonstrated to play an important role in adverse cardiac remodeling and fibrosis. However, the mechanisms of U II-induced myocardial fibrosis remain to be clarified. We postulated that U II alters transforming growth factor-beta1 (TGF-beta1) expression, and thereby modulates cardiac fibroblast collagen metabolism. Experiments were conducted using cardiac fibroblast from neonatal Wistar rats to determine the expression of TGF-beta1, and the role of U II receptor UT in this process. The functional role of TGF-beta1 and UT in modulating U II effects on type I, III collagen mRNA expression and 3H-proline incorporation was also analyzed. TGF-beta1 gene and protein expression were consistently identified in quiescent cardiac fibroblasts. U II increased the expression of TGF-beta1 mRNA and protein in a time-dependent manner. This effect was UT mediated, because UT antagonist urantide abolished U II-induced TGF-beta1 expression. U II-induced increase in type I, III collagen mRNA expression and 3H-proline incorporation were both inhibited by a specific TGF-beta1 neutralizing antibody and UT receptor antagonist urantide. Hence, our results indicate that TGF-beta1 is upregulated in cardiac fibroblasts by U II via UT and modulates profibrotic effects of U II. These findings provide novel insights into U II-induced cardiac remodeling.     

Human endothelial nitric oxide synthase gene delivery protects against cardiac remodeling and reduces oxidative stress after myocardial infarction

Nitric oxide (NO) has been shown to play a key role in the regulation of cardiac hypertrophy and fibrosis in response to myocardial ischemia in part by antagonizing the action of angiotensin II (Ang II). In this study, we investigated the potential protective role of human endothelial nitric oxide synthase (eNOS) in left ventricular (LV) remodeling after myocardial infarction (MI) by a somatic gene transfer approach. Male Wistar rats underwent coronary artery ligation to induce MI. One week after surgery, adenovirus encoding the human eNOS or luciferase gene under the control of the CMV promoter/enhancer was injected into rats via the tail vein, and animals were sacrificed at 1 and 5 weeks after gene transfer. Successful gene transfer was evaluated based on increased levels of NO and cGMP in the heart, measured at one week after eNOS gene delivery. Six weeks after MI, the LV end-diastolic pressure, heart weight, LV axis length and cardiomyocyte size were markedly increased compared to the Sham group, while eNOS gene delivery significantly reduced these parameters. Rats receiving control virus developed considerably more fibrotic lesions identified by Sirius Red staining and collagen I immunostaining compared to Sham rats, and eNOS gene delivery significantly reduced collagen accumulation. eNOS gene transfer also reduced TUNEL-positive apoptotic cells. The cardioprotective effect of NO was accompanied by reduced NADH and NADPH oxidase activities and superoxide formation, TGF-beta1 and p27 levels, JNK activation, NF-kappa B nuclear translocation, and caspase-3 activity. This study shows that NO may play an important role in attenuating cardiac remodeling and apoptosis after myocardial infarction via suppression of oxidative stress-mediated signaling pathways.     

Functional role and species-specific contribution of arginases in pulmonary fibrosis

Lung fibrosis is characterized by increased deposition of ECM, especially collagens, and enhanced proliferation of fibroblasts. l-arginine is a key precursor of nitric oxide, asymmetric dimethylarginine, and proline, an amino acid enriched in collagen. We hypothesized that l-arginine metabolism is altered in pulmonary fibrosis, ultimately affecting collagen synthesis. Expression analysis of key enzymes in the arginine pathway, protein arginine methyltransferases (Prmt), arginine transporters, and arginases by quantitative (q) RT-PCR and Western blot revealed significant upregulation of arginase-1 and -2, but not Prmt or arginine transporters, during bleomycin-induced pulmonary fibrosis in mice. HPLC revealed a concomitant, time-dependent decrease in pulmonary l-arginine levels. Arginase-1 and -2 mRNA and protein expression was increased in primary fibroblasts isolated from bleomycin-treated mice, compared with controls, and assessed by qRT-PCR and Western blot analysis. TGF-beta1, a key profibrotic mediator, induced arginase-1 and -2 mRNA expression in primary and NIH/3T3 fibroblasts. Treatment of fibroblasts with the arginase inhibitor, NG-hydroxy-l-arginine, attenuated TGF-beta1-stimulated collagen deposition, but not collagen mRNA expression or Smad signaling, in fibroblasts. In human lungs derived from patients with idiopathic pulmonary fibrosis, arginase activity was unchanged, but arginase-1 expression significantly decreased when compared with donor lungs. Our results thus demonstrate that arginase-1 is expressed and functionally important for collagen deposition in lung fibroblasts. TGF-beta1-induced upregulation of arginase-1 suggests an interplay between profibrotic agents and l-arginine metabolism during the course of lung fibrosis in the mouse, whereas species-specific regulatory mechanisms may account for the differences observed in mouse and human.     

Connective Tissue Growth Factor Overexpression in Cardiomyocytes Promotes Cardiac Hypertrophy and Protection against Pressure Overload

Connective tissue growth factor (CTGF) is a secreted protein that is strongly induced in human and experimental heart failure. CTGF is said to be profibrotic; however, the precise function of CTGF is unclear. We generated transgenic mice and rats with cardiomyocyte-specific CTGF overexpression (CTGF-TG). To investigate CTGF as a fibrosis inducer, we performed morphological and gene expression analyses of CTGF-TG mice and rat hearts under basal conditions and after stimulation with angiotensin II (Ang II) or isoproterenol, respectively. Surprisingly, cardiac tissues of both models did not show increased fibrosis or enhanced gene expression of fibrotic markers. In contrast to controls, Ang II treated CTGF-TG mice displayed preserved cardiac function. However, CTGF-TG mice developed age-dependent cardiac dysfunction at the age of 7 months. CTGF related heart failure was associated with Akt and JNK activation, but not with the induction of natriuretic peptides. Furthermore, cardiomyocytes from CTGF-TG mice showed unaffected cellular contractility and an increased Ca²⁺ reuptake from sarcoplasmatic reticulum. In an ischemia/reperfusion model CTGF-TG hearts did not differ from controls.Our data suggest that CTGF itself does not induce cardiac fibrosis. Moreover, it is involved in hypertrophy induction and cellular remodeling depending on the cardiac stress stimulus. Our new transgenic animals are valuable models for reconsideration of CTGF''s profibrotic function in the heart.     

DIOL Triterpenes Block Profibrotic Effects of Angiotensin II and Protect from Cardiac Hypertrophy

Background

The natural triterpenes, erythrodiol and uvaol, exert anti-inflammatory, vasorelaxing and anti-proliferative effects. Angiotensin II is a well-known profibrotic and proliferative agent that participates in the cardiac remodeling associated with different pathological situations through the stimulation and proliferation of cardiac fibroblasts. Therefore, the aim of the study was to investigate the preventive effects of the natural triterpenes erythrodiol and uvaol on the proliferation and collagen production induced by angiotensin II in cardiac myofibroblasts. Their actions on cardiac hypertrophy triggered by angiotensin II were also studied.

Methodology/Principal Findings

The effect of erythrodiol and uvaol on angiotensin II-induced proliferation was evaluated in cardiac myofibroblasts from adult rats in the presence or the absence of the inhibitors of PPAR-γ, GW9662 or JNK, SP600125. The effect on collagen levels induced by angiotensin II was evaluated in cardiac myofibroblasts and mouse heart. The presence of low doses of both triterpenes reduced the proliferation of cardiac myofibroblasts induced by angiotensin II. Pretreatment with GW9662 reversed the effect elicited by both triterpenes while SP600125 did not modify it. Both triterpenes at high doses produced an increase in annexing-V binding in the presence or absence of angiotensin II, which was reduced by either SP600125 or GW9662. Erythrodiol and uvaol decreased collagen I and galectin 3 levels induced by angiotensin II in cardiac myofribroblasts. Finally, cardiac hypertrophy, ventricular remodeling, fibrosis, and increases in myocyte area and brain natriuretic peptide levels observed in angiotensin II-infused mice were reduced in triterpene-treated animals.

Conclusions/Significance

Erythrodiol and uvaol reduce cardiac hypertrophy and left ventricle remodeling induced by angiotensin II in mice by diminishing fibrosis and myocyte area. They also modulate growth and survival of cardiac myofibroblasts. They inhibit the angiotensin II-induced proliferation in a PPAR-γ-dependent manner, while at high doses they activate pathways of programmed cell death that are dependent on JNK and PPAR-γ.     

Differential response of human cardiac fibroblasts to angiotensin I and angiotensin II

The vasoactive peptide angiotensin II (Ang II) has been implicated as a mediator of myocardial fibrosis. We carried out a comparative investigation of the effects of Ang II and its precursor Ang I on collagen metabolism and proliferation in cultured human cardiac fibroblasts. Cardiac fibroblasts responded to both Ang I and Ang II with concentration-dependent increases in collagen synthesis but no proliferation. The stimulatory effect of Ang II was abolished by the AT(1) receptor antagonist losartan but not the AT(2) receptor antagonist PD123319. The response to Ang I was not affected by either antagonist, nor by the angiotensin-converting enzyme (ACE) inhibitor captopril. In conclusion, Both Ang I and Ang II stimulate collagen synthesis of human cardiac fibroblasts, the effect of Ang II occurring via the AT(1) receptor whilst Ang I appears to exert a direct effect through non-Ang II-dependent mechanisms. These results suggest distinct roles for angiotensin peptides in the development of cardiac fibrosis.     

Diabetes-induced upregulation of urotensin II and its receptor plays an important role in TGF-beta1-mediated renal fibrosis and dysfunction

Urotensin II (UII) was identified as the ligand for a novel G protein-coupled receptor, GPR14. UII was found not only to have a potent vasoconstrictive action but also to have profibrotic effects in the heart. The present study was to define whether UII and GPR14 also play important roles in diabetes-induced renal fibrosis and dysfunction. Diabetic rats were induced using streptozotocin, and the rat proximal tubular epithelial cells (NRK-52E) were used for the in vitro mechanism study. Results showed that expression of UII and GPR14 was significantly upregulated at both mRNA and protein levels in the diabetic kidneys compared with controls. The upregulated expressions of UII and GPR14 in the kidney were accompanied by significant increases in the renal profibrotic factor transforming growth factor (TGF)-beta1 expression, the renal extracellular matrix (fibronectin and collagen IV) accumulation, and the renal dysfunction (increases in urinal N-acetyl-beta-d-glucosaminidase content, 24-h urinary retinol-binding protein excretion rate, and decrease in creatinine clearance rate). Exposure of NRK-52E cells to 10(-8) mol/l UII for 48 h caused a significant increase of TGF-beta1, but not ANG II, production that was GPR14- and calcium-dependent, since GPR14 small-interfering RNA and calcium channel blocker nimodipine or calcium chelator EDTA all could abolish the induction of TGF- beta1 by UII. Furthermore, exposure of NRK-52E cells to TGF-beta1 or ANG II also increased UII and GPR14 mRNA expressions. These results suggested that diabetes-induced upregulation of UII and GPR14, most likely through autocrine and/or paracrine mechanisms, plays an important role in TGF-beta1-mediated renal fibrosis and dysfunction.     

Mast cells are required for the development of renal fibrosis in the rodent unilateral ureteral obstruction model

Mast cells are associated with inflammation and fibrosis. Whether they protect against or contribute to renal fibrosis is unclear. Based on our previous findings that mast cells can express and secrete active renin, and that angiotensin (ANG II) is profibrotic, we hypothesized that mast cells play a critical role in tubulointerstitial fibrosis. We tested this hypothesis in the 14-day unilateral ureteral obstruction (UUO) model in rats and mast cell-deficient (MCD) mice (WBB6F1-W/Wv) and their congenic controls (CC). In the 14-day UUO rat kidney, mast cell number is increased and they express active renin. Stabilizing mast cells in vivo with administration of cromolyn sodium attenuated the development of tubulointerstitial fibrosis, which was confirmed by measuring newly synthesized pepsin-soluble collagen and blind scoring of fixed trichrome-stained kidney sections accompanied by spectral analysis. Fibrosis was absent in UUO kidneys from MCD mice unlike that observed in the CC mice. Losartan treatment reduced the fibrosis in the CC UUO kidneys. The effects of mast cell degranulation and renin release were tested in the isolated, perfused kidney preparation. Mast cell degranulation led to renin-dependent protracted flow recovery. This demonstrates that mast cell renin is active in situ and the ensuing ANG II can modulate intrarenal vascular resistance in the UUO kidney. Collectively, the data demonstrate that mast cells are critical to the development of renal fibrosis in the 14-day UUO kidney. Since renin is present in human kidney mast cells, our work identifies potential targets in the treatment of renal fibrosis.     

Angiotensin converting enzyme-2 is protective but downregulated in human and experimental lung fibrosis

Earlier work from this laboratory showed that local generation of angiotensin (ANG) II is required for the pathogenesis of experimental pulmonary fibrosis and that ANG peptides are expressed robustly in the lungs of patients with idiopathic pulmonary fibrosis (IPF). Angiotensin converting enzyme-2 (ACE-2) degrades the octapeptide ANG II to form the heptapeptide ANG1-7 and thereby limits ANG II accumulation. On this basis, we hypothesized that ACE-2 would be protective against experimental lung fibrogenesis and might be downregulated in human and experimental lung fibrosis. In lung biopsy specimens from patients with IPF, ACE-2 mRNA and enzyme activity were decreased by 92% (P<0.01) and 74% (P<0.05), respectively. ACE-2 mRNA and activity were also decreased similarly in the lungs of bleomycin-treated rats and C57-BL6 mice. In mice exposed to low doses of bleomycin, lung collagen accumulation was enhanced by intratracheal administration of either ACE-2-specific small interfering RNAs (siRNAs) or the peptide DX(600), a competitive inhibitor of ACE-2 (P<0.05). Administration of either ACE-2 siRNA or DX(600) significantly increased the ANG II content of mouse lung tissue above the level induced by bleomycin alone. Coadministration of the ANG II receptor antagonist saralasin blocked the DX(600)-induced increase in lung collagen. Moreover, purified recombinant human ACE-2, delivered to mice systemically by osmotic minipump, attenuated bleomycin-induced lung collagen accumulation. Together, these data show that ACE-2 mRNA and activity are severely downregulated in both human and experimental lung fibrosis and suggest that ACE-2 protects against lung fibrogenesis by limiting the local accumulation of the profibrotic peptide ANG II.     

Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells.

Protein tyrosine phosphorylation has not been considered to be important for cellular activation by phospholipase C-linked vasoactive peptides. We found that endothelin, angiotensin II, and vasopressin (AVP), peptides that signal via phospholipase C activation, rapidly enhanced tyrosine phosphorylation of proteins of approximate molecular mass 225, 190, 135, 120, and 70 kDa in rat renal mesangial cells. The phosphorylated proteins were cytosolic or membrane-associated, and none were integral to the membrane, suggesting that the peptide receptors are not phosphorylated on tyrosine. Epidermal growth factor (EGF), which does not activate phospholipase C in these cells, induced the tyrosine phosphorylation of its own 175-kDa receptor, in addition to five proteins of identical molecular mass to those phosphorylated in response to endothelin, AVP, and angiotensin II. This suggests that in mesangial cells there is a common signaling pathway for phospholipase C-coupled agonists and agonists classically assumed to signal via receptor tyrosine kinase pathways, such as EGF. The phorbol ester, phorbol 12-myristate 13-acetate, and the synthetic diacylglycerol, oleoyl acetylglycerol, stimulated the tyrosine phosphorylation of proteins identical to those phosphorylated by the phospholipase C-linked peptides, suggesting that protein kinase C (PKC) activation is sufficient to active tyrosine phosphorylation. However, the PKC inhibitor, staurosporine, and down-regulation of PKC activity by prolonged exposure to phorbol esters completely inhibited tyrosine phosphorylation in response to PMA but not to endothelin, AVP, or EGF. In conclusion, endothelin, angiotensin II, and AVP enhances protein tyrosine phosphorylation via at least two pathways, PKC-dependent and PKC-independent. Although activation of PKC may be sufficient to enhance protein tyrosine phosphorylation, PKC is not necessary and may not be the primary route by which these agents act. At least one of these pathways is shared with the growth factor EGF, suggesting not only common intermediates in the signaling pathways for growth factors and vasoactive peptides but also perhaps common cellular tyrosine kinases which phosphorylate these intermediates.     

Elevated formation of pyridinoline cross-links by profibrotic cytokines is associated with enhanced lysyl hydroxylase 2b levels

The hallmark of fibrosis is the excessive accumulation of collagen. The deposited collagen contains increased pyridinoline cross-link levels due to an overhydroxylation of lysine residues within the collagen telopeptides. Lysyl hydroxylase 2b (LH2b) is the only lysyl hydroxylase consistently up-regulated in several forms of fibrosis, suggesting that an enhanced LH2b level is responsible for the overhydroxylation of collagen telopeptides. The present paper reports the effect of profibrotic cytokines on the expression of collagen, lysyl hydroxylases and lysyl oxidase in normal human skin fibroblasts, as well as the effect on pyridinoline formation in the deposited matrix. All three isoforms of TGF-beta induce a substantial increase in LH2b mRNA levels, also when expressed relatively to the mRNA levels of collagen type I alpha2 (COL1A2). The TGF-beta isoforms also clearly influence the collagen cross-linking pathway, since higher levels of pyridinoline cross-links were measured. Similar stimulatory effects on LH2b/COL1A2 mRNA expression and pyridinoline formation were observed for IL-4, activin A, and TNF-alpha. An exception was BMP-2, which has no effect on LH2b/COL1A2 mRNA levels nor on pyridinoline formation. Our data show for the first time that two processes, i.e., up-regulation of LH2b mRNA levels and increased formation of pyridinoline cross-links, previously recognized to be inherent to fibrotic processes, are induced by various profibrotic cytokines.     

Transforming growth factor-beta1 stimulates vascular endothelial growth factor 164 via mitogen-activated protein kinase kinase 3-p38alpha and p38delta mitogen-activated protein kinase-dependent pathway in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix synthesis leading to progressive glomerular fibrosis. The intracellular signaling mechanisms involved in this process remain incompletely understood. The p38 mitogen-activated protein kinase (MAPK) is a major stress signal transducing pathway that is rapidly activated by TGF-beta1 in mesangial cells. We have previously demonstrated MKK3 as the immediate upstream MAPK kinase required for selective activation of p38 MAPK isoforms, p38alpha and p38delta, and stimulation of pro-alpha1(I) collagen by TGF-beta1 in murine mesangial cells. In this study, we further sought to determine MAPK kinase 3 (MKK3)-dependent TGF-beta1 responses by gene expression profiling analysis utilizing mesangial cells isolated from Mkk3-/- mice compared with Mkk3+/+ controls. Interestingly, vascular endothelial growth factor (VEGF) was identified as a TGF-beta1-induced gene affected by deletion of Mkk3. VEGF is a well known endothelial mitogen, whose actions in nonendothelial cell types are still not well understood. We confirmed that TGF-beta1 increased VEGF mRNA and protein synthesis of VEGF164 and VEGF188 isoforms in wild-type mesangial cells. However, in the Mkk3-/- mesangial cells, both TGF-beta1-induced VEGF mRNA and VEGF164 protein expression were inhibited, whereas TGF-beta1-induced VEGF188 protein expression was unaffected. Furthermore, transfection of dominant negative mutants of p38alpha and p38delta resulted in marked inhibition of TGF-beta1-induced VEGF164 expression but not VEGF188, and treatment with recombinant mouse VEGF164 increased collagen and fibronectin mRNA expression in mesangial cells. Taken together, our findings suggest a critical role for the MKK3-p38alpha and p38delta MAPK pathway in mediating VEGF164 isoform-specific stimulation by TGF-beta1 in mesangial cells. Further, VEGF164 stimulates collagen and fibronectin expression in mesangial cells and thus in turn enhances TGF-beta1-induced extracellular matrix and may play an important role in progressive glomerular fibrosis.