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Modulation of microvascular smooth muscle adhesion and mechanotransduction by integrin-linked kinase.

ABSTRACT: In this study, we investigated the involvement of integrin-linked kinase (ILK) in the adhesion of arteriolar vascular smooth muscle cells (VSMC) to fibronectin (FN) and in the mechano-responsiveness of VSMC focal adhesions (FA).ILK was visualized in VSMC by expressing EGFP-ILK and it was knocked down using ILK-shRNA constructs. Atomic force microscopy (AFM) was used to characterize VSMC interactions with FN, VSMC stiffness and to apply and measure forces at a VSMC single FA site.ILK was localized to FA and silencing ILK promoted cell spreading, enhanced cell adhesion, reduced cell proliferation and reduced downstream phosphorylation of GSK-3beta and PKB/Akt. AFM studies demonstrated that silencing ILK enhanced alpha5beta1 integrin adhesion to FN and enhanced VSMC contraction in response to a pulling force applied at the level of a single FN-FA site.ILK functions in arteriolar VSMC appear linked to multiple signaling pathways and processes that inhibit cell spreading, cell adhesion, FA formation, adhesion to FN and the mechano-responsiveness of FN-FA sites.

SUBMITTER: Huang S

PROVIDER: S-EPMC2923833 | biostudies-other | 2010 Feb

REPOSITORIES: biostudies-other

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Modulation of microvascular smooth muscle adhesion and mechanotransduction by integrin-linked kinase.

Huang Shaoxing S Sun Zhe Z Li Zhaohui Z Martinez-Lemus Luis A LA Meininger Gerald A GA

Microcirculation (New York, N.Y. : 1994) 20100201 2

<h4>Objective</h4>In this study, we investigated the involvement of integrin-linked kinase (ILK) in the adhesion of arteriolar vascular smooth muscle cells (VSMC) to fibronectin (FN) and in the mechano-responsiveness of VSMC focal adhesions (FA).<h4>Methods</h4>ILK was visualized in VSMC by expressing EGFP-ILK and it was knocked down using ILK-shRNA constructs. Atomic force microscopy (AFM) was used to characterize VSMC interactions with FN, VSMC stiffness and to apply and measure forces at a VS ...[more]

PMID: 20163538

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Project description:Recombinant osteopontin (rOPN) has been reported to be neuroprotective in stroke animal models. The purpose of this study is to investigate a potential role and mechanism of nasal administration of rOPN on preserving the vascular smooth muscle phenotype in early brain injury after subarachnoid hemorrhage (SAH).One hundred and ninety-two male adult Sprague-Dawley rats were used. The SAH model was induced by endovascular perforation. Integrin-linked kinase small interfering RNA was intracerebroventricularly injected 48 hours before SAH. The integrin receptor antagonist fibronectin-derived peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), focal adhesion kinase inhibitor Fib-14, and Rac-1 inhibitor NSC23766 were administered 1 hour before SAH induction. rOPN was administered via the intracerebroventricular and nasal route after SAH. SAH grade, neurological scores, brain water content, brain swelling, hematoxylin and eosin staining, India ink angiography, Western blots, and immunofluorescence were used to study the mechanisms of rOPN on the vascular smooth muscle phenotypic transformation.The marker proteins of vascular smooth muscle phenotypic transformation ?-smooth muscle actin decreased and embryonic smooth muscle myosin heavy chain (SMemb) increased significantly at 24 and 72 hours in the cerebral arteries after SAH. rOPN prevented the changes of ?-smooth muscle actin and SMemb and significantly alleviated neurobehavioral dysfunction, increased the cross-sectional area and the lumen diameter of the cerebral arteries, reduced the brain water content and brain swelling, and improved the wall thickness of cerebral arteries. These effects of rOPN were abolished by GRGDSP, integrin-linked kinase small interfering RNA, and NSC23766. Intranasal application of rOPN at 3 hours after SAH also reduced neurological deficits.rOPN prevented the vascular smooth muscle phenotypic transformation and improved the neurological outcome, which was possibly mediated by the integrin receptor/integrin-linked kinase/Rac-1 pathway.

Project description:BACKGROUND: Diabetic nephropathy (DN) is the leading cause of chronic kidney disease and is associated with excessive cardiovascular morbidity and mortality. The angiotensin converting enzyme inhibitor (ACEI) benazepril has been shown to slow the progression of chronic renal disease and have beneficial effects in patients with a combination of chronic renal disease and cardiovascular disease. Transforming growth factor-?(1) (TGF-?(1)) plays a central role in the pathogenesis and progression of DN. Integrin-linked kinase (ILK) can modulate TGF-?(1)-induced glomerular mesangial cell (GMC) injury, which is a prominent characteristic of renal pathology in kidney diseases. As an integrin cytoplasmic-binding protein, ILK regulates fibronectin (FN) matrix deposition and the actin cytoskeleton. Smooth muscle ?-actin (?-SMA) is involved in progressive renal dysfunction in both human and experimental renal disease. METHODS: To explore the mechanisms of benazepril's reno-protective effects, we examined the expression of TGF-?(1), ILK, and ?-SMA in GMC exposed to high glucose (HG) and in the kidneys of streptozotocin (STZ)-induced diabetic rats using real-time quantitative RT-PCR and western blot analysis. To elucidate the mechanism(s) of the effect of benazepril on GMC cellular processes, we assessed the effect of benazepril on Angiotensin II (Ang II) signalling pathways using western blot analysis. RESULTS: The expression of TGF-?(1), ILK, and ?-SMA increased significantly in the diabetic group compared with the control group. Benazepril treatment inhibited the expression of these genes in DN but failed to rescue the same levels in the control group. Similar results were found in GMC treated with HG or benazepril. Ang II increased ERK and Akt phosphorylation in the HG group, and benazepril could not completely block these responses, suggesting that other molecules might be involved in the progression of DN. Our findings suggest that benazepril decreases ILK and ?-SMA expression, at least in part, by affecting the interactions between Ang II and TGF-?(1). CONCLUSIONS: The findings described here support the hypothesis that the HG milieu of diabetes increases TGF-?(1) secretion, which increases the synthesis of ILK and ?-SMA that are involved in the progression of DN. This might be an important mechanism of the benazepril renal-protective function in the pathogenesis of DN.

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Modulation of microvascular smooth muscle adhesion and mechanotransduction by integrin-linked kinase.

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Modulation of microvascular smooth muscle adhesion and mechanotransduction by integrin-linked kinase.

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