Mohammed El Mabrouk

Structure, Endothelial Function, Cell Growth, and Inflammation in Blood Vessels of Angiotensin II–Infused Rats Role of Peroxisome Proliferator–Activated Receptor-γ

Background—Pioglitazone and rosiglitazone, thiazolidinedione peroxisome proliferator–activated receptor-&ggr; (PPAR&ggr;) activators, reduce blood pressure (BP) in some hypertensive models by unclear mechanisms. We tested the hypothesis that pioglitazone or rosiglitazone would prevent BP elevation and vascular dysfunction in angiotensin (Ang) II–infused rats by direct vascular effects. Methods and Results—Sprague-Dawley rats received Ang II (120 ng · kg−1 · min−1 SC) with or without pioglitazone (10 mg · kg−1 · d−1) or rosiglitazone (5 mg · kg−1 · d−1) for 7 days. Systolic BP, elevated in Ang II–infused rats (176±5 mm Hg) versus controls (109±2 mm Hg, P <0.01), was reduced by pioglitazone (134±2 mm Hg) or rosiglitazone (123±2 mm Hg). In mesenteric small arteries studied in a pressurized myograph, media/lumen ratio was increased (P <0.05) and acetylcholine-induced relaxation impaired in Ang II–infused rats (P <0.05); both were normalized by the thiazolidinediones. In Ang II–infused rats, vascular DNA synthesis (by 3H-thymidine incorporation); expression of cell cycle proteins cyclin D1 and cdk4, angiotensin II type 1 receptors, vascular cell adhesion molecule-1, and platelet and endothelial cell adhesion molecule; and nuclear factor-&kgr;B activity were increased. These changes were abrogated by pioglitazone or rosiglitazone. Conclusions—Thiazolidinedione PPAR-&ggr; activators attenuated the development of hypertension, corrected structural abnormalities, normalized cell growth, and improved endothelial dysfunction induced by Ang II and prevented upregulation of angiotensin II type 1 receptors, cell cycle proteins, and proinflammatory mediators. Thiazolidinediones may be useful in the prevention and/or treatment of hypertension, particularly when it is associated with insulin resistance or diabetes mellitus.

Src is an important mediator of extracellular signal-regulated kinase 1/2-dependent growth signaling by angiotensin II in smooth muscle cells from resistance arteries of hypertensive patients

Abstract—The role of c-Src in growth signaling by angiotensin (Ang) II was investigated in vascular smooth muscle cells (VSMCs) from arteries of hypertensive patients. c-Src and extracellular signal–regulated kinase 1/2 (ERK1/2) activity, proto-oncogene expression, activating protein-1 (AP-1) DNA-binding activity, and DNA and protein synthesis were studied in Ang II–stimulated VSMCs derived from small peripheral resistance arteries of normotensive subjects (NTs, n=5) and age-matched untreated hypertensive patients (HTs, n=10). Ang II type 1 (AT1) and type 2 (AT2) receptor status was also assessed. Ang II dose-dependently increased the synthesis of DNA and protein, with enhanced effects in VSMCs from HTs. PD 098,059, a selective inhibitor of the ERK1/2 pathway, attenuated Ang II–stimulated growth in HTs. The effects of PD 098,059 were greater in HTs than in NTs. In NTs, Ang II transiently increased ERK1/2 phosphorylation, whereas in HTs, Ang II–stimulated actions were augmented and sustained. PP2, a selective Src inhibitor, reduced ERK1/2 activity and normalized ERK1/2 responses in HTs. Ang II–induced c-Src phosphorylation was 2- to 3-fold greater in HTs than in NTs. In HTs but not NTs, kinase activation was followed by overexpression of c-fos and enhanced AP-1 DNA-binding activity. PD 098,059 and PP2 attenuated these responses. AT1 receptor expression was similar in NTs and HTs. In HT cells transfected with c-fos antisense oligodeoxynucleotide, Ang II–stimulated growth was reduced compared with sense oligodeoxynucleotide. Our findings suggest that augmented Ang II–stimulated VSMC growth is mediated via hyperactivation of c-Src–regulated ERK1/2-dependent pathways, leading to overexpression of c-fos mRNA and enhanced AP-1 DNA-binding activity. Because AT1 receptor expression was unaltered in HTs, increased Ang II signaling may be a postreceptor phenomenon. These data define a signal transduction pathway whereby Ang II mediates exaggerated growth in VSMCs from HTs.

p38 MAP Kinase Regulates Vascular Smooth Muscle Cell Collagen Synthesis by Angiotensin II in SHR But Not in WKY

Vascular remodeling in hypertension is associated with cell growth and increased deposition of extracellular matrix components, particularly collagen. Mechanisms underlying these processes are unclear, but MAP kinases, particularly ERK1/2 and p38 MAP kinase, may be important. We studied the role of ERK1/2 and p38 MAP kinase in vascular smooth muscle cell (VSMC) collagen synthesis and growth mediated by angiotensin (Ang) II in spontaneously hypertensive rats (SHR). Cultured mesenteric VSMC from Wistar-Kyoto rats and SHR were used. Phosphorylation of ERK1/2 and p38 MAP kinase were assessed by Western blots with phosphospecific antibodies. Ang II–stimulated DNA and collagen synthesis were determined by measuring incorporation of 3H-thymidine and 3H-proline, respectively. mRNA expression of procollagen I and III was determined by reverse transcription–polymerase chain reaction. Ang II increased ERK1/2 and p38 MAP kinase phosphorylation. Responses were augmented in SHR. Effects were inhibited by irbesartan, a selective AT1 antagonist, but not by PD123319, a selective AT2 blocker. Ang II stimulated 3H-thymidine and 3H-proline incorporation. These actions were enhanced 2- to 3-fold in SHR. PD98059, selective inhibitor of the ERK1/2 pathway, attenuated Ang II–induced growth and collagen effects and normalized responses in SHR. SB212190, a selective p38 MAP kinase inhibitor, did not alter Ang II–elicited DNA synthesis but reduced collagen production and mRNA expression of procollagen I and III in SHR. These data demonstrate that (1) Ang II–mediated activation of p38 and ERK1/2 is increased in SHR, (2) augmented growth responses are generated by ERK1/2-dependent, p38 MAP kinase-independent pathways, and (3) p38 MAP kinase influences Ang II–induced collagen production in SHR but not in Wistar-Kyoto rats. These results indicate differential roles of ERK1/2 and p38 MAP kinase in AT1-stimulated VSMC growth and collagen production, which may contribute to vascular remodeling in hypertension.

Differential activation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen activated-protein kinase by AT1 receptors in vascular smooth muscle cells from Wistar-Kyoto rats and spontaneously hypertensive rats.

Objectives The present study investigates effects of angiotensin II on activation of extracellular signal-regulated protein kinase (ERK) 1/2, p38 mitogen activated-protein kinase (p38MAPK) and c-Jun amino terminal kinase (JNK) in vascular smooth muscle cells from spontaneously hypertensive rats (SHR). Methods Vascular smooth muscle cells (VSMC) from mesenteric arteries of Wistar–Kyoto (WKY) rats and SHR were studied. Angiotensin II-induced phosphorylation of ERK1/2, JNK and p38MAPK were assessed by Western blot analysis. c-fos mRNA expression by angiotensin II was determined by reverse transcriptase-polymerase chain reaction in the absence and presence of PD98059, selective inhibitor of ERK1/2-dependent pathways and SB202190, selective p38MAPK inhibitor. Results Angiotensin II increased phosphorylation of ERK1/2 and p38MAPK, but not JNK. Responses were significantly increased in SHR compared with WKY. Irbesartan, AT1 receptor antagonist, but not PD123319, AT2 receptor blocker, abolished angiotensin II-induced effects. PP2, selective Src inhibitor, decreased angiotensin II-mediated activation of MAP kinases. Angiotensin II increased c-fos mRNA expression in SHR and had a small stimulatory effect in WKY. These actions were inhibited by PD98059, whereas SB202190 had no effect. Conclusions Angiotensin II-induced activation of vascular ERK1/2 and p38MAPK is increased in SHR. These effects are mediated via AT1 receptors, which activate Src-dependent pathways. Overexpression of c-fos mRNA in SHR is due to ERK1/2-dependent, p38MAPK-independent pathways. Our results suggest that angiotensin II activates numerous MAP kinases in VSMCs and that differential activation of these kinases may be important in altered growth signaling in VSMCs from SHR.

TGF-β-induced expression of tissue inhibitor of metalloproteinases-3 gene in chondrocytes is mediated by extracellular signal-regulated kinase pathway and Sp1 transcription factor

Transforming growth factor (TGF‐β1) is a potent inducer of chondrogenesis and stimulant of cartilage extracellular matrix (ECM) synthesis. Tissue inhibitor of metalloproteinases‐3 (TIMP‐3) is located in ECM and is the major inhibitor of matrix metalloproteinases (MMPs) and aggrecanase, the principal enzymes implicated in collagen and aggrecan degradation in arthritis. We investigated the role of extracellular‐signal‐regulated kinase (ERK)‐mitogen‐activated protein kinases (MAPK) and Sp1 transcription factor in TGF‐β‐induced TIMP‐3 gene in chondrocytes and chondrosarcoma cells. TGF‐β time‐dependently induced a sustained phosphorylation of ERK‐MAPKs in primary human or bovine chondrocytes. Inhibitors of this pathway, PD98059 and U0126, downregulated TGF‐β‐induced expression of TIMP‐3 RNA and protein. Since the ERKs can phosphorylate Sp1, and the promoter of human TIMP‐3 gene contains four Sp1‐binding sites, we investigated whether Sp1 is a downstream target of this pathway. Mithramycin and WP631, the agents that prevent binding of Sp1 to its consensus site, downregulated TGF‐β‐inducible TIMP‐3 expression. Indeed, mithramycin blocked TGF‐β‐stimulated Sp1 binding activity. Transfection of cytomegalovirus (CMV) promoter‐Sp1 plasmid increased TIMP‐3 promoter (−940 to +376)‐driven luciferase activity. Depletion of Sp1 by transfection of an antisense phosphorothioate oligonucleotide suppressed TGF‐β‐induced TIMP‐3 protein expression, while its sense homolog had no effect. These results suggest that activation of ERK‐MAPK pathway and Sp1 transcription factor play a pivotal role in the induction of TIMP‐3 by TGF‐β in chondrocytes.

Interleukin-1 induction of aggrecanase gene expression in human articular chondrocytes is mediated by mitogen-activated protein kinases.

Background/Aims: We investigated the unknown molecular mechanisms of Interleukin-1 (IL-1β)-induced cartilage aggrecan degeneration by aggrecanase (ADAMTS-A Disintegrin And Metalloproteinase with ThromboSpondin motifs) in human articular chondrocytes, a model mimicking human arthritis. Methods: Chondrocytes were pretreated with various pharmacological inhibitors and then stimulated with IL-1β for 24 h. ADAMTS-4 expression or activity was studied by RT-PCR or ELISA and other proteins measured by Western blotting. Results: MAP kinase kinase-specific inhibitor, U0126 inhibited IL-1-induced phosphorylation of ERK1/2 and down-regulated ADAMTS-4 expression and activity. Protein 38 inhibitor, SB203580 down-regulated the phosphorylation of p38 and its target, activating transcription factor-2 (ATF-2), ADAMTS-4 mRNA and activity. C-Jun N-terminal kinase (JNK) inhibitor, SP600125 diminished IL-1-stimulated JNK phosphorylation, ADAMTS-4 mRNA expression and enzyme activity. A c-fos/lipoxygenase pathway inhibitor and antioxidant, nordihydroguaiaretic acid (NDGA) significantly suppressed ADAMTS-4 mRNA induction and activity. Activating protein (AP-1) and nuclear factor kappa B (NF-ĸB) transcription factor inhibitors, curcumin and pyrrolidine dithiocarbamate (PDTC) partially inhibited ADAMTS-4 induction and activity. Conclusion: These results suggest partial involvement of ERK-, p38-and JNK-MAPKs as well as AP-1, ATF-2 and NF-ĸB transcription factors in IL-1-induced ADAMTS-4 in chondrocytes. Inhibition of these targets by the specific pharmacological agents could be useful for reducing aggrecanase-driven cartilage resorption in arthritis.

Antiproliferative effect of L-NAME on rat vascular smooth muscle cells

The nitric oxide synthase (NOS) inhibitor L-NAME may have growth inhibitory effects in vivo. We investigated in vitro the potential growth inhibitory effects of three different NOS inhibitors: L-NAME (1 mM), LNMMA (1 mM) and aminoguanidine (0.5 mM), on fetal bovine serum (FBS) and platelet derived growth factor (PDGF-BB)-stimulated growth in cultured vascular smooth muscle cells (VSMCs). [3H]-thymidine incorporation into rat mesenteric VSMCs was measured as an index of VSMCs proliferation (DNA synthesis) and activation of extracellular signal regulated kinase (ERK1/2), a major signaling event in cell growth, was measured by western blot assay. PDGF-BB (0-5 ng/mL) and FBS (0-5%) increased [3H]-thymidine incorporation in a dose-dependent manner up to 6-10 fold. L-NAME significantly reduced PDGF-BB (5 ng/ml) and FBS (5%) stimulated DNA synthesis by 46% and 38% respectively. The increase of [3H]-thymidine incorporation induced by PDGF-BB and FBS was unaltered by L-NMMA. In contrast, aminoguanidine induced an increase in FBS and PDGF-BB-stimulated [3H]-thymidine incorporation of 64% and 34% respectively above cells not exposed to aminoguanidine. ERK1/2 phosphorylation induced by PDGF-BB and FBS was not affected by pre-treatment with L-NAME or aminoguanidine. In conclusion, NOS inhibitors differentially influence DNA synthesis in VSMCs: L-NAME inhibits FBS and PDGF-BB-stimulated cellular proliferation whereas aminoguanidine accentuates FBS and PDGF-BB-stimulated VSMCs proliferation. These phenomena are independent of the ERK1/2 pathway. The growth inhibitory effects of L-NAME may be related to differences in properties from other NOS inhibitors, and independent of its ability to inhibit NOS.

Expression of Cell Cycle Proteins in Blood Vessels of Angiotensin II–Infused Rats Role of AT1 Receptors

Angiotensin II is an important modulator of cell growth through AT1 receptors, as demonstrated both in vivo and in vitro. We investigated the role of proteins involved in the cell cycle, including cyclin D1, cyclin-dependent kinase 4 (cdk4), and cyclin-dependent kinase inhibitors p21 and p27 in blood vessels of angiotensin II–infused rats and the effect therein of the AT1-receptor antagonist losartan. Male Sprague-Dawley rats were infused for 7 days with angiotensin II (120 ng/kg per minute SC) and/or treated with losartan (10 mg/kg per day orally). DNA synthesis in mesenteric arteries was evaluated by radiolabeled 3H-thymidine incorporation. The expression of cyclin D1, cdk4, p21, and p27, which play critical roles during the G1-phase of the cell cycle process, was examined by Western blot analysis. Tail-cuff systolic blood pressure (mm Hg) was elevated (P <0.01, n=9) in angiotensin II–infused rats (161.3±8.2) versus control rats (110.1±5.3) and normalized by losartan (104.4±3.2). Radiolabeled 3H-thymidine incorporation (cpm/100 &mgr;g DNA) showed that angiotensin II infusion significantly increased DNA synthesis (152±5% versus 102±6% of control rats, P <0.05). Expression of cyclin D1 and cdk4 was significantly increased in the angiotensin II group to 213.7±8% and 263.6±37% of control animals, respectively, whereas expression of p21 and p27 was significantly decreased in the angiotensin II group to 23.2±10.4% and 10.3±5.3% of control animals, respectively. These effects induced by angiotensin II were normalized in the presence of losartan. Thus, when AT1 receptors are stimulated in vivo, DNA synthesis is enhanced in blood vessels by activation of cyclin D1 and cdk4. Reduction in cell cycle kinase inhibitors p21 and p27 may contribute to activation of growth induced by in vivo AT1 receptor stimulation.

Interleukin‐4 antagonizes oncostatin M and transforming growth factor beta‐induced responses in articular chondrocytes

Oncostatin M (OSM) stimulates cartilage degradation in rheumatoid arthritis (RA) by inducing matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS; a disintegrin and metalloproteinase with thrombospondin motif). Transforming growth factor beta (TGF‐β1) induces cartilage repair in joints but in excessive amounts, promotes inflammation. OSM and TGF‐β1 also induce tissue inhibitor of metalloproteinase‐3 (TIMP‐3), an important natural inhibitor of MMPs, aggrecanases, and tumor necrosis factor alpha converting enzyme (TACE), the principal proteases involved in arthritic inflammation and cartilage degradation. We studied cartilage protective mechanisms of the antiinflammatory cytokine, interleukin‐4 (IL‐4). IL‐4 strongly (MMP‐13 and TIMP‐3) or minimally (ADAMTS‐4) suppressed OSM‐induced gene expression in chondrocytes. IL‐4 did not affect OSM‐stimulated phosphorylation of extracellular signal‐regulated kinases (ERKs), protein 38 (p38), c‐Jun N‐terminal kinase (JNK) and Stat1. Lack of additional suppression with their inhibitors suggested that MMP‐13, ADAMTS‐4, and TIMP‐3 inhibition was independent of these mediators. IL‐4 also downregulated TGF‐β1‐induced TIMP‐3 gene expression, Smad2, and JNK phosphorylation. Additional suppression of TIMP‐3 RNA by JNK inhibitor suggests JNK implication. The cartilage protective effects of IL‐4 in animal models of arthritis may be due to its inhibition of MMPs and ADAMTS‐4 expression. However, suppression of TIMP‐3 suggests caution for using IL‐4 as a cartilage protective therapy. J. Cell. Biochem. 103: 588–597, 2008.

Enhanced Expression of Tissue Inhibitor of Metalloproteinases-4 Gene in Human Osteoarthritic Synovial Membranes and Its Differential Regulation by Cytokines in Chondrocytes

Objective: Tissue inhibitors of metalloproteinases (TIMPs) are multi-functional proteins with matrix metalloproteinases-inhibiting activities. We studied expression of anti-inflammatory, TIMP-4 gene in human joint tissues and its regulation by arthritis-associated cytokines. Results: TIMP-4 RNA expression originating from synovial fibroblasts was significantly (2.4 fold; p<0.001) elevated in 8 osteoarthritic (OA) versus 7 non-arthritic synovial membranes. Non-arthritic and OA femoral head and knee chondrocytes displayed substantial but variably constitutive expression of the TIMP-4 mRNA. In articular chondrocytes, transforming growth factor beta (TGF-β1) and oncostatin M (OSM) upregulated TIMP-4 RNA and protein expression while interleukin-1 (IL-1β) and tumor necrosis factor alpha (TNF-α) did not, suggesting differential regulation by arthritis-associated cytokines. Interleukin 17 (IL-17) mildly induced TIMP-4 mRNA. TGF-β1 induction of TIMP-4 expression was partly inhibited by ERK pathway and Sp1 transcription factor inhibitors. Conclusion: Enhanced TIMP-4 gene expression in OA synovial membranes and cartilage may be due to induction by TGF-β1, OSM and IL-17, suggesting its pathophysiological role in tissue remodeling in human joints. TGF-β1 induction of TIMP-4 expression is mediated partly by ERK pathway and Sp1 transcription factor.