Role of elevated EGFR phosphorylation in the induction of structural remodelling and altered mechanical properties of resistance artery from type 2 diabetic mice.

BACKGROUND: Type 2 diabetes is associated with microvascular complications. We hypothesized that the sustained elevated EGFR phosphorylation produces structural wall remodelling and altered mechanical properties of mesenteric resistance artery (MRA) in type 2 diabetes. METHODS: Freshly isolated MRA (80-100 microm diameter) from type 2 diabetic (db(-)/db(-), diabetic) and non-diabetic (db(-)/db(+), control) mice were subjected to pressure-passive diameter and wall thickness relationships; western blot analysis and immunohistology. RESULTS: Data indicated that MRA from diabetic mice have a smaller passive diameter than MRA from control mice under intra-luminal pressure range from 25 to 125 mmHg. Measurements of wall thickness : lumen diameter ratios (21 +/- 1.8 vs 14 +/- 1.2 at 75 mmHg diabetic vs control, respectively), wall thickness and remodelling index (38 +/- 5% vs control) revealed eutrophic structural remodelling of MRA from diabetic mice, which was strengthened with histology. Mechanical properties revealed a great strain-stress relationship in MRA from control versus diabetic mice indicating increased stiffness in MRA from diabetic mice. Western blot analysis showed increased collagen type 1 content in a freshly isolated MRA from the type 2 diabetic mice when compared to control mice. Diabetic mice treated with EGFR inhibitor (AG1478, 10 mg/kg/day) for 2 weeks showed reduced EGFR phosphorylation, wall thickness, collagen type 1 content, and improved the altered mechanical properties of MRA. CONCLUSION: These data provide evidence regarding the role of EGFR in morphological wall remodelling and altered mechanical properties of MRA from type 2 diabetic mice. This may identify new therapeutic targets for the control of vascular structure and therefore have important implications in type 2 diabetes.

Role of advanced glycation end products with oxidative stress in resistance artery dysfunction in type 2 diabetic mice.

OBJECTIVE: Type 2 diabetes is associated with increased advanced glycation end product (AGE) formation and vasculopathy. We hypothesized that AGEs contribute to resistance artery dysfunction. METHODS AND RESULTS: Type 2 diabetic db(-)/db(-) (diabetic) and nondiabetic db(-)/db(+) (control) mice were treated with the AGE inhibitor (aminoguanidine: 50 mg/Kg/d) for 3 months. Isolated mesenteric resistance arteries (MRAs) were mounted in an arteriograph. Pressure-induced myogenic tone (MT) was increased in diabetic mice but was unaffected by aminoguanidine treatment. Phenylephrine-induced contraction and nitric oxide donor-induced endothelium-independent relaxation were similar in all groups. In diabetic mice, endothelium-dependent relaxation in response to shear-stress or acetylcholine was altered and was associated with reduced eNOS protein and mRNA expression. Aminoguanidine treatment improved endothelial function and restored eNOS expression. AGE formation and hypoxia markers (plasminogen activator inhibitor 1 and Bnip3) were increased in MRA from diabetic mice and normalized with Aminoguanidine. Primary cultured endothelial cells (ECs) isolated from resistance arteries subjected to high glucose for 48 hours showed decreased eNOS expression and phosphorylation in response to calcium ionophore. High glucose decreased antioxidant protein (MnSOD) and increased prooxidant proteins (gp91phox) expression leading to increased oxidative stress generation, as assessed by DHE staining and endothelial NADH/NADPH oxidase activity. The preincubation of ECs with aminoguanidine restored eNOS-phosphorylation and expression as well as the balance between pro- and antioxidant factors induced by high glucose. CONCLUSIONS: We provide evidence of a link between AGEs, oxidative stress, and resistance artery EC dysfunction in type 2 diabetic mice. Thus, AGEs and oxidative stress may be a potential target for overcoming diabetic microvessels complications.

Microvessel vascular smooth muscle cells contribute to collagen type I deposition through ERK1/2 MAP kinase, alphavbeta3-integrin, and TGF-beta1 in response to ANG II and high glucose.

This study determines that vascular smooth muscle cell (VSMC) signaling through extracellular signal-regulated kinase (ERK) 1/2-mitogen-activated protein (MAP) kinase, alphavbeta(3)-integrin, and transforming growth factor (TGF)-beta1 dictates collagen type I network induction in mesenteric resistance arteries (MRA) from type 1 diabetic (streptozotocin) or hypertensive (HT; ANG II) mice. Isolated MRA were subjected to a pressure-passive-diameter relationship. To delineate cell types and mechanisms, cultured VSMC were prepared from MRA and stimulated with ANG II (100 nM) and high glucose (HG, 22 mM). Pressure-passive-diameter relationship reduction was associated with increased collagen type I deposition in MRA from HT and diabetic mice compared with control. Treatment of HT and diabetic mice with neutralizing TGF-beta1 antibody reduced MRA stiffness and collagen type I deposition. Cultured VSMC stimulated with HG or ANG II for 5 min increased ERK1/2-MAP kinase phosphorylation, whereas a 48-h stimulation induced latent TGF-beta1, alphavbeta(3)-integrin, and collagen type 1 release in the conditioned media. TGF-beta1 bioactivity and Smad2 phosphorylation were alphavbeta(3)-integrin-dependent, since beta(3)-integrin antibody and alphavbeta(3)-integrin inhibitor (SB-223245, 10 microM) significantly prevented TGF-beta1 bioactivity and Smad2 phosphorylation. Pretreatment of VSMC with ERK1/2-MAP kinase inhibitor (U-0126, 1 microM) reduced alphavbeta(3)-integrin, TGF-beta1, and collagen type 1 content. Additionally, alphavbeta(3)-integrin antibody, SB-223245, TGF-beta1-small-intefering RNA (siRNA), and Smad2-siRNA (40 nM) prevented collagen type I network formation in response to ANG II and HG. Together, these data provide evidence that resistance artery fibrosis in type 1 diabetes and hypertension is a consequence of abnormal collagen type I release by VSMC and involves ERK1/2, alphavbeta(3)-integrin, and TGF-beta1 signaling. This pathway could be a potential target for overcoming small artery complications in diabetes and hypertension.

Elevated epidermal growth factor receptor phosphorylation induces resistance artery dysfunction in diabetic db/db mice.

OBJECTIVE: We previously showed epidermal growth factor receptor (EGFR) transactivation to be key mechanism in the regulation of resistance artery myogenic tone. Type 2 diabetes is associated with microvascular complications. We hypothesized that elevated EGFR phosphorylation contributes to resistance artery dysfunction in type 2 diabetes. RESEARCH DESIGN AND METHODS AND RESULTS: Diabetic db/db and nondiabetic (control) mice were treated with EGFR inhibitor (AG1478; 10 mg x kg(-1) x day(-1)) for 2 weeks. Isolated coronary artery and mesenteric resistance artery (MRA) were mounted in an arteriograph. Pressure-induced myogenic tone was increased in MRA and coronary artery from diabetic mice and normalized by AG1478. Phenylephrine-induced contraction and nitric oxide donor-induced relaxation were similar in all groups. Endothelium-dependent relaxation in response to shear stress and acetylcholine of MRA and coronary artery from diabetic mice was altered and associated with reduced endothelial nitric oxide synthase (eNOS) expression and phosphorylation. Treated diabetic mice with AG1478 improved coronary artery and MRA endothelial function and restored eNOS expression. Immunostaining and Western blot analysis showed increased endothelial and smooth muscle cell EGFR phosphorylation of MRA and coronary artery from diabetic mouse, which was reduced by AG1478. Primary cultured endothelial cells from resistance arteries treated with high glucose for 48 h showed an increase of EGFR phosphorylation associated with eNOS expression and phosphorylation decrease in response to calcium ionophore. Pretreatment of endothelial cells with AG1478 prevented the effect of high glucose. CONCLUSIONS: This study provides evidence of the role of elevated EGFR phosphorylation in coronary artery and MRA dysfunction in diabetic db/db mice. Therefore, EGFR should be a potential target for overcoming diabetic small artery complications.

Role of ACE/AT2R complex in the control of mesenteric resistance artery contraction induced by ACE/AT1R complex activation in response to Ang I.

OBJECTIVES: In this study, we will determine the function of the interaction between AT2R and ACE, and AT1R and ACE in the control of mesenteric resistance artery (MRA) tone from normotensive (NT) and Angiotensin II (AII)-dependent hypertensive (HT) mice. METHODS-RESULTS: Hypertension was induced by infusion of Ang-II (200 ng/kg/day) for 3 weeks. Freshly MRA (100-120 microm) were isolated from HT and NT mice and mounted in an arteriograph. Dose-response of Ang-I induced a similar contraction of MRA from NT and HT mice, which was increased after endothelium removal. AT2R antagonist (PD123319, 1 microM) significantly increased Ang-I-induced contraction of MRA from NT but not from HT mice. In addition, PD123319 significantly increased in vivo blood pressure in response to Ang-I. Luminal incubation with ACE-antibody (50 ng/ml) to block only endothelial ACE function significantly enhanced Ang-I-induced contraction of MRA from NT mice. ACE inhibitor (captopril, 10 microM) completely blocked Ang-I-induced contraction of MRA from both animals and prevented the increased blood pressure. Freshly isolated MRA subjected to immunoprecipitation, Western blot analysis and RT-PCR revealed AT1R/ACE and AT2R/ACE complexes formation, and similar AT1R, AT2R, and ACE expression level in both groups. CONCLUSION: The present findings show the existence of ACE/AT2R and ACE/AT1R complexes on endothelial cells and VSMC, respectively. ACE/AT2R complex plays a modulator effect on ACE/AT1R-SMC-induced contraction of MRA, which is altered in hypertension.

Mice lacking the gene encoding for MMP-9 and resistance artery reactivity.

OBJECTIVES: To define the link between the deletion of gene encoding for metalloproteinase 9 and resistance artery reactivity, we studied in vitro smooth muscle and endothelial cell function in response to pressure, shear stress, and pharmacological agents. BACKGROUND: Matrix metalloproteinases play a crucial role in the regulation of extracellular matrix turnover and structural artery wall remodeling. METHODS: Resistance arteries were isolated from mice lacking gene encoding for MMP-9 (KO) and their control (WT). Hemodynamic, pharmacology approaches, and Western blot analysis were used in this study. RESULTS: The measurement of blood pressure in vivo was similar in KO and WT mice. Pressure-induced myogenic tone, contractions to angiotensin-II and phenylephrine were similar in both groups. The inhibition of MMP2/9 ((2R)-2-[(4-biphenylylsulfonyl) amino]-3-phenylpropionic acid) significantly decreased myogenic tone in WT and had no effect in KO mice. Relaxation endothelium-dependent (flow-induced- dilation 41.3+/-0.6 vs. 21+/-1.6 at 10 microl/min in KO and WT mice, respectively, P<0.05) and eNOS expression were increased in KO compared to WT mice. The inhibition of eNOS with L-NAME significantly decreased endothelium response to shear stress, which was more pronounced in KO mice resistance arteries (-26.83+/-2.5 vs. -15.84+/-2.3 at 10 microl/min in KO and WT, respectively, P<0.05). However, the relaxation to exogenous nitric oxide-donor was similar in both groups. CONCLUSION: Our study provides evidence of a selective effect of MMP-9 on endothelium function. Thus, MMP-9 gene deletion specifically increased resistance artery dilation endothelium-dependent and eNOS expression. Based on our results, MMP-9 could be a potential therapeutic target in cardiovascular disease associated with resistance arteries dysfunction.

Hydrogen peroxide acts as relaxing factor in human vascular smooth muscle cells independent of map-kinase and nitric oxide.

We previously showed that hydrogen peroxide (H2O2) induced resistance artery relaxation independent of endothelium. Thus, in this study we investigated the mechanism of relaxation induced by H2O2 on human renal vascular smooth muscle cell (HVSMC). HVSMC were stimulated with H2O2 and/or angiotensin II (Ang II), proline-rich-tyrosine-kinase-2 (PYK2), ERK1/2 MAP-Kinase, and myosin light chain 20 phosphorylation (Lc20) were assessed using Western blot analysis in the presence of potassium channel blockers, MAP-Kinase, and nitric oxide synthesis (NOS) inhibitors. H2O2 increased PYK2 and ERK1/2 phosphorylation, and at the same time decreased Lc20 phosphorylation. AngII increased phosphorylation of PYK2, ERK1/2 and Lc20, whereas, the pretreatment of HVSMC with H2O2 decreased Lc20 phosphorylation induced by AngII. MEK inhibition, decreased ERK1/2 phosphorylation, but had no effect on the inhibition of phosphorylation of Lc20 induced by H2O2. The inhibition of Ca2(+)-dependent K+ channels (BKCa) and NOS did not block the decrease of Lc20 phosphorylation in response to H2O2. On the other hand, pretreatment of HVSMC with 60 mM of KCl, increased rather than decreased Lc20 phosphorylation in response to H2O2. This study shows the evidence that H2O2 acts as a relaxing factor and as an activator of PYK2 and ERK1/2 in Human renal VSMC. The relaxation induced by H2O2 is independent of BKCa, ERK1/2 MAP-Kinase and NOS pathways. The relaxing effect to H2O2 changes to contracting effect when the potassium channels are compromised.

Role of SHP-1, Kv.1.2, and cGMP in nitric oxide-induced ERK1/2 MAP kinase dephosphorylation in rat vascular smooth muscle cells.

OBJECTIVE: Nitric oxide (NO) elicits relaxation in vascular smooth muscle cells (VSMC) that is associated with guanylate cyclase (GC) and K(+) channel activation. In this study we determined the mechanisms that lead to ERK1/2 MAP kinase dephosphorylation in response to NO. METHODS: VSMC were treated with the NO donor SNAP or sodium nitroprusside (SNP), and ERK1/2, Src homology (SH) 1 domain-containing protein tyrosine phosphatase (SHP-1), and Kv.1.2 phosphorylation were assessed by immunoprecipitation and Western blot analysis. RESULTS: NO decreased basal ERK1/2 phosphorylation in a dose- and time-dependent manner. NO-induced ERK1/2 dephosphorylation was detected at 1 min and sustained for 30 min. Pre-treatment with the GC inhibitor ODQ or the protein tyrosine phosphatase inhibitor I prevented ERK1/2 dephosphorylation induced by SNAP. The inhibition of protein phosphatase 1A/2A had no effect on ERK1/2 dephosphorylation induced by SNAP. Treatment with cromakalim A, a nonspecific K(+) channel activator, also induced ERK1/2 dephosphorylation, while blockade of Kv.1.2 K(+) channels (AM92016 hydrochloride) prevented NO-induced ERK1/2 dephosphorylation. In addition, SNAP induced SHP-1 phosphorylation, and the Kv.1.2 dephosphorylation increase and SHP-1 phosphorylation was blocked by ODQ or AM92016. The basal interaction between ERK1/2 and SHP-1 was decreased in response to SNAP stimulation. SHP-1 also interacted with Kv.1.2 under basal conditions and participates in Kv.1.2 activation. Using the mouse mesenteric resistance artery, we found that ERK1/2 MAP kinase is involved in regulation of myogenic tone. CONCLUSION: Thus, our study provides the first evidence that NO controls basal ERK1/2 phosphorylation by a signaling cascade that involves a dynamic signaling complex between cGMP, Kv.1.2 and SHP-1.