TBX3, a downstream target of TGF-ß1, inhibits mesangial cell apoptosis.

Chronic kidney disease (CKD) is an increasingly common condition characterized by progressive loss of functional nephrons leading to renal failure. TGF-ß1-induced mesangial cell (MC) phenotype alterations have been linked to the genesis of CKD. Here we show that TGF-ß1 regulates TBX3 gene expression in MC. This gene encodes for two main isoforms, TBX3.1 and TBX3+2α. TBX3.1 has been implicated in cell immortalization, proliferation and apoptosis by inhibiting p14(ARF)-Mdm2-p53 pathway, while TBX3+2α role has not been defined. We demonstrated that TBX3 overexpression abrogated MC apoptosis induced by serum deprivation. Moreover, we observed an enhancement in TBX3 protein expression both in glomerular and tubular regions in the model of 5/6 nephrectomy, temporally related to increased expression of TGF-ß1, type IV collagen and fibronectin. Our results indicate that TBX3 acts as an anti-apoptotic factor in MC in vitro and may be involved in the mechanism by which TGF-ß1 induces glomerulosclerosis and tubular fibrosis during the progression of nephropathies.

Targeting bone morphogenetic protein signaling on renal and vascular diseases.

PURPOSE OF REVIEW: Normal development and adult physiology of the kidney and vasculature rely heavily on bone morphogenetic proteins (BMPs). Here we compile evidence that favors the notion that BMPs are also critically involved in the process of generation and maintenance of renal and vascular diseases. RECENT FINDINGS: Molecular manipulation of BMP signaling in vivo and in vitro has been instrumental in showing the protective role of BMPs on renal fibrosis and diabetic nephropathy. Similarly, activation of those pathways produces phenotypic changes in vascular smooth muscle and endothelial cells, tightly linked to the pathogenesis of vascular calcification, hypertrophy and atherosclerosis. SUMMARY: Gain-of-function and loss-of-function experiments targeting BMP pathway agonists and inhibitors lead to significant progress in the comprehension of renal and vascular normal and altered behavior. The demonstration that BMP signaling plays an important part in pathological conditions of the vasculature and the kidney opens up possibilities for the development of diagnostic and therapeutic tools.

Gremlin promotes vascular smooth muscle cell proliferation and migration.

Recent reports highlight the importance of BMP in the vasculature. We investigated the expression pattern and role of the BMP antagonist gremlin in VSMC. We detected gremlin mRNA constitutive expression in adult and embryonic rat aortic VSMC, and in rat carotids. In vitro analysis demonstrated that angiotensin II, TGF-beta1 and PDGF induced significant changes in gremlin mRNA expression. Gremlin stable overexpression in A7r5 cells blocked BMP signaling. BMP-induced reduction in VSMC DNA synthesis was markedly inhibited by gremlin overexpression. In fact, gremlin overexpression increased DNA synthesis and cell counts, and accelerated cell cycle progression of VSMC, through mechanisms that include p27(kip1) down-regulation. Gremlin also led to marked increments in VSMC migration. In addition, gremlin gene silencing promoted a significant blockade on cell proliferation and migration. In vivo studies disclosed increased gremlin protein expression in the neointima of balloon-injured carotid arteries. In summary, the BMP antagonist gremlin is constitutively expressed in the normal vasculature. Gremlin induces VSMC proliferation and migration and is significantly regulated by growth factors and injury. We postulate that gremlin plays a part in the development of pathological phenotypic changes of adult VSMC.

Determinants of Notch-3 receptor expression and signaling in vascular smooth muscle cells: implications in cell-cycle regulation.

The Notch family of receptors and ligands plays an important role in cell fate determination, vasculogenesis, and organogenesis. Mutations of the Notch-3 receptor result in an arteriopathy that predisposes to early-onset stroke. However, the functional role of the Notch signaling pathway in adult vascular smooth muscle cells (VSMCs) is poorly characterized. This study documents that the Notch-3 receptor, the ligand Jagged-1, and the downstream transcription factor, HESR-1, are expressed in the normal adult rat carotid artery, and that this expression is modulated after vascular injury. In cultured VSMCs, both angiotensin II and platelet-derived growth factor (PDGF) markedly downregulated Notch-3 and Jagged-1 through ERK-dependent signaling mechanisms and prevented the glycosylation of Jagged-1. The downregulation of Jagged-1 and Notch-3 was associated with a decrease in CBF-1-mediated gene transcription activation and a fall in the mRNA levels of the downstream target transcription factor HESR-1. To test the hypothesis that the Notch pathway was coupled to growth regulation, we generated VSMC lines overexpressing the constitutively active form of Notch-3 (A7r5-N3IC). These cells exhibited a biphasic growth behavior in which the growth rate was retarded during the subconfluent phase and failed to decelerate at postconfluence. The lack of cell-cycle arrest in postconfluent A7r5-N3IC was associated with an attenuated upregulation of the cell-cycle inhibitor p27(kip) relative to control cells. This study documents the regulation of the Jagged-1 and Notch-3 genes in VSMCs by growth factor stimulation as well as a role for Notch-3 as a determinant of VSMC growth.

DNA microarray profiling to identify angiotensin-responsive genes in vascular smooth muscle cells: potential mediators of vascular disease.

Angiotensin II (Ang II) induces changes in vessel structure by its capacity to activate genes that are coupled to signaling pathways such as extracellular signal-regulated kinase (ERK), p38, and phosphatidylinositol 3-kinase (PI3K). Using a DNA microarray containing 5088 genes and expressed sequence tags, we initially established a database of replicated experiments (n=4) to define the variances in mRNA expression in response to Ang II versus vehicle treatment. We observed a wide range of values for the coefficients of variation in a gene-specific manner. Guided by power calculations, we used statistical inference on a sufficient number of experimental replicates to minimize the number of false-negatives and define a subset of Ang II-responsive genes (P<0.05). To further characterize the molecular circuitry that couples Ang II stimulation with mRNA expression, we assessed expression profiles in the presence and absence of inhibitors of ERK, p38, and PI3K. Using two different methods of computational cluster analysis, we identified a subset of six matricellular proteins (eg, osteopontin and plasminogen activator inhibitor-1) that are coordinately upregulated by Ang II via an ERK/p38-dependent pathway. In addition, these cluster analyses identified calpactins I and II as novel Ang II-responsive genes. Given that Ang II promotes vascular lesion formation, we examined whether this matricellular gene cluster was also coordinately regulated in vivo. Indeed, we demonstrate that both calpactin I and osteopontin are upregulated in response to vascular injury. Taken together, the combined use of DNA microarrays, statistical inference, and cluster analysis identified novel, coordinately regulated Ang II-responsive genes that may mediate vascular lesion formation.

Increasing doses of simvastatin versus combined ezetimibe/simvastatin: effect on circulating endothelial progenitor cells.

BACKGROUND: Patients with coronary artery disease (CAD) should be treated with statins to attain very low cholesterol levels, in order to reduce cardiovascular adverse events. More than 70% of these patients do not reach the appropriate cholesterol goal despite moderate statin doses. However, it is not known whether therapeutic uptitration with different lipid-lowering strategies has a similar "pleiotropic" effect on atherosclerotic endothelial dysfunction evaluated by measurement of endothelial progenitor cells (EPCs). OBJECTIVE: We sought to compare, in patients with stable CAD and with a low-density lipoprotein cholesterol (LDL-C) >70 mg/dL on treatment with simvastatin 20 mg, the effects on EPCs by increasing simvastatin to 80 mg versus adding ezetimibe 10 mg. METHODS: Patients (n = 68, 63 ± 9 years, 39% men) were randomly allocated to receive ezetimibe 10/simvastatin 20 mg or simvastatin 80 mg for 6 weeks. Circulating EPCs were measured by flow cytometry before and after the treatment. RESULTS: Both strategies presented similar effects on metabolic parameters. The LDLs were equally reduced by ezetimibe 10/simvastatin 20 mg and simvastatin 80 mg (28.9% ± 13% vs 21.1% ± 33%; P = .46, respectively). The levels of EPCs were unaffected by ezetimibe 10/simvastatin 20 mg (median [25th, 75th]: pre- vs posttreatment, 7.0 [2.3; 13.3] vs 3.1 [0.1; 13.2] EPCs/10(4) mononuclear cells; P = .43) or simvastatin 80 mg (pre- vs posttreatment, 6.1 [2.9; 15.2] vs 4.0 [1.4; 10.7] EPCs/10(4) mononuclear cells; P = .5), and there were no differences between the groups on treatment effects (P = .9). CONCLUSIONS: Among stable patients with CAD and with an LDL-C >70 mg/dL on simvastatin 20 mg, increasing simvastatin dose to 80 mg or adding ezetimibe 10 mg promoted similar further cholesterol reduction but did not have incremental effects on circulating EPCs. These data suggest that the effects of simvastatin moderate doses on EPCs are not increased by intensive lipid-lowering strategies (clinicaltrials.gov: NCT00474123).

Pleiotropic effects of ezetimibe/simvastatin vs. high dose simvastatin.

BACKGROUND: In the setting of stable coronary artery disease (CAD), it is not known if the pleiotropic effects of cholesterol reduction differ between combined ezetimibe/simvastatin and high-dose simvastatin alone. OBJECTIVE: We sought to compare the anti-inflammatory and antiplatelet effects of ezetimibe 10mg/simvastatin 20mg (E10/S20) with simvastatin 80 mg (S80). METHODS AND RESULTS: CAD patients (n=83, 63 ± 9 years, 57% men) receiving S20, were randomly allocated to receive E10/S20 or S80, for 6 weeks. Lipids, inflammatory markers (C-reactive protein, interleukin-6, monocyte chemoattractant protein-1, soluble CD40 ligand and oxidized LDL), and platelet aggregation (platelet function analyzer [PFA]-100) changes were determined. Baseline lipids, inflammatory markers and PFA-100 were similar between groups. After treatment, E10/S20 and S80 patients presented, respectively: (1) similar reduction in LDL-C (29 ± 13% vs. 28 ± 30%, p=0.46), apo-B (18 ± 17% vs. 22 ± 15%, p=0.22) and oxidized LDL (15 ± 33% vs. 18 ± 47%, p=0.30); (2) no changes in inflammatory markers; and, (3) a higher increase of the PFA-100 with E10/S20 than with S80 (27 ± 43% vs. 8 ± 33%, p=0.02). CONCLUSIONS: These data suggest that among stable CAD patients treated with S20, (1) both E10/S20 and S80 were equally effective in further reducing LDL-C; (2) neither treatment had any further significant anti-inflammatory effects; and (3) E10/S20 was more effective than S80 in inhibiting platelet aggregation. Thus, despite similar lipid lowering and doses 4× less of simvastatin, E10/S20 induced a greater platelet inhibitory effect than S80.

Tormentic acid reduces vascular smooth muscle cell proliferation and survival.

The triterpene tormentic acid (TA) has been reported to exhibit anticancer, anti-inflammatory and anti-atherogenic properties, and minimal toxicity has been detected in in vivo. Vascular smooth muscle cell (VSMC) proliferation and apoptosis resistance are hallmarks of vasculoproliferative diseases, such as post-angioplasty restenosis. The present study was designed to assess the effects of TA on the phenotype of cultured VSMC. The exposure of VSMC to TA (30 muM) significantly increased apoptosis of serum-deprived A7r5 cells, whereas cell survival in the presence of 10% fetal bovine serum was less affected by the drug. On the other hand, even in the presence of serum, A7r5 cell proliferation was significantly inhibited by TA, an effect that persisted for at least 8 days of daily administration of TA. As preservation of endothelial integrity is critical to normal vascular function, we also evaluated the effects of TA on human umbilical cord endothelial cells (HUVEC). Interestingly, TA did not produce significant changes in the levels of apoptosis and proliferation of HUVEC. Our data indicate that TA is a VSMC apoptosis inducer and proliferation inhibitor. The anti-growth action in VSMC in the presence of serum, and the absence of significant effects in endothelial cells suggest that TA may control VSMC abnormal proliferation and cell death resistance without affecting the normal vasculature. We conclude that TA should be investigated further as a potential tool for the prevention and treatment of proliferative vascular diseases, particularly in the setting of post-angioplasty restenosis.

Orally administered rapamycin does not modify rat aortic vascular tone.

Rapamycin (RP; rapamune, sirolimus) is a potent inhibitor of vascular smooth muscle cell proliferation and migration. RP was demonstrated to reduce vascular neointimal formation in different animal models of vascular smooth muscle cell proliferation, and clinical use of RP-eluting stents promotes significant reductions in in-stent restenosis rates. However, high costs still preclude the widespread use of these devices. Oral administration of RP associated to bare metal stent delivery has been advocated as an effective and considerably less expensive alternative for restenosis prevention. It is noteworthy that the presence of RP-eluting stents has been associated with reduced endothelial-dependent vasodilation and coronary spasm. In addition, RP has been demonstrated to prevent vasculogenesis. This study evaluated the effects of RP on endothelium-dependent vascular tone and demonstrated that in vitro incubation with high concentrations of RP did not modify either contraction or relaxation of aortic rings. Similar results were obtained after in vivo administration of the drug. These findings suggest that function of adult, non-proliferative rat endothelial cells involved in vascular tone control is not affected by orally administered RP.

Bradykinin induces a calcium-store- dependent calcium influx in mouse mesangial cells.

Bradykinin (BK) elicits extracellular-dependent [Ca2+](i) elevations in mouse mesangial cells (MMC) that are not blocked by verapamil, nifedipine, L-nicardipine, NiCl(2), or LaCl(3). The aim of the present study was to evaluate the mechanisms involved in calcium influx induced by BK in MMC. [Ca2+](i) was analyzed through spectrofluorometry employing fura-2-AM, and the data were expressed as [Ca2+](i )obtained/[Ca2+](i )basal ratio. Heparin (IP(3), a receptor antagonist) almost abolished the effects of BK in MMC (1.85 +/- 0.15 vs. 1.13 +/- 0.02, n = 4, p = 0.001). Following external and intracellular calcium store depletion, BK's effect was absent even after successful extracellular calcium replenishment. ML-7 (a myosin light chain kinase inhibitor) blocked responses to thapsigargin (2.62 +/- 0.13 vs. 1.11 +/- 0.04, n = 4, p < 0.001), but not those of BK (6.51 +/- 0.39, n = 6, vs. 5.86 +/- 1.17, n = 4, p = 0.39). On the other hand, genistein (a tyrosine kinase inhibitor) was able to inhibit thapsigargin (3.12 +/- 0.22, n = 5, vs. 1.28 +/- 0.16, n = 4, p < 0.001) as well as BK responses (6.46 +/- 0.66 vs. 2.89 +/- 0.61, n = 4, p < 0.05). Econazole (a P-450 monooxygenase inhibitor) inhibited the responses to both thapsigargin (3.45 +/- 0.16 vs. 1.03 +/- 0.03, n = 4, p < 0.001) and BK (6.49 +/- 0.83, n = 6, vs. 1.17 +/- 0.08, n = 4, p = 0.01). Finally, responses to BK were not affected by indomethacin (6.69 +/- 0.66 vs. 6.57 +/- 0.87, n = 4, p = 0.916). Thus, BK promotes an IP(3)-sensitive store-dependent calcium influx in MMC. This phenomenon seems to involve tyrosine kinase and P-450 monooxygenase products in its transduction pathway.

Amitriptyline eliminates calculi through urinary tract smooth muscle relaxation.

BACKGROUND: We investigated the effects of amitriptyline in the urinary tract smooth muscle and urolithiasis. METHODS: Cats presenting with obstructive acute renal failure (ARF) received amitriptyline, and renal function and survival rates were analyzed. Isometric contractions and membrane potentials of rat, pig, or human isolated urinary tract smooth muscle were recorded in the presence or absence of amitriptyline. RESULTS: Twenty cats with obstructive ARF caused by urethral plugs received amitriptyline. In all cases, plugs were completely eliminated, and renal function returned to normal, with a 100% survival rate in the follow-up. Amitriptyline produced potent relaxations in rat urethral strips, accompanied by significant reductions in urethral ring membrane potential. This effect was prevented by pretreatment of urethral rings with 4-aminopyridine (4-AP), a voltage-dependent potassium channel blocker. Amitriptyline abolished in a reversible manner acetylcholine-, bradykinin-, and KCl-induced contractions in rat isolated bladder, and this effect was also prevented by 4-AP. Of interest, spontaneous and KCl-induced contractions of pig and human isolated ureter were also blocked by amitriptyline. CONCLUSION: Our results indicate that amitriptyline is an effective and potent relaxant of urinary tract smooth muscle and this effect is mediated by opening of voltage dependent-potassium channels. We suggest that amitriptyline administration may help to promote elimination of urinary calculi.

Coordinate Notch3-hairy-related transcription factor pathway regulation in response to arterial injury. Mediator role of platelet-derived growth factor and ERK.

The Notch family of receptors and downstream effectors plays a critical role in cell fate determination during vascular ontogeny. Moreover, the human cerebral autosomal dominant artriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) syndrome of premature stroke and dementia is a heritable arteriopathy with alterations in vascular smooth muscle cells (VSMCs) resulting from mutations within Notch3. However, the expression and regulation of the Notch and hairy-related transcription factor (HRT) pathway in adult VSMCs in vitro and in vivo remain poorly characterized. The present study documents that the well-described modulation of VSMC fate in response to vascular injury and growth factor activation involves a coordinate regulation of the Notch and HRT pathways. Furthermore, platelet-derived growth factor promotes a similar coordinate down-regulation of the Notch receptors and HRT genes in cultured VSMCs via an ERK-dependent signaling pathway. Moreover, we established that HRT1 and HRT2 are direct downstream target genes of Notch3 signaling in VSMCs and determined that the activity of the nuclear protein RBP-Jk is essential for their regulation. These findings provide initial insight into the context- and cell type-dependent coordinate regulation of Notch3 and downstream HRT transcriptional pathway effector genes in VSMCs in vitro and in vivo that may have important implications for understanding the role of Notch signaling in human health and vascular disease.