Aging induces a progressive decline in vasoconstrictor responses in central and peripheral arteries. This study investigated the hypothesis that vascular smooth muscle (VSM) contractile function declines with age in soleus muscle feed arteries (SFA). Contractile function of cannulated SFA isolated from young (4 months) and old (24 months) Fischer 344 rats was assessed by measuring constrictor responses of denuded (endothelium removed) SFA to norepinephrine (NE), phenylephrine (PE), and angiotensin II (Ang II). In addition, we investigated the role of RhoA signaling in modulation of VSM contractile function. Structural and functional characteristics of VSM cells were evaluated by fluorescence imaging and atomic force microscopy (AFM). Results indicated that constrictor responses to PE and Ang II were significantly impaired in old SFA, whereas constrictor responses to NE were preserved. In the presence of a Rho-kinase inhibitor (Y27632), constrictor responses to NE, Ang II, and PE were significantly reduced in young and old SFA. In addition, the age-group difference in constrictor responses to Ang II was eliminated. ROCK1 and ROCK2 content was similar in young and old VSM cells, whereas pROCK1 and pROCK2 were significantly elevated in old VSM cells. Aging was associated with a reduction in smooth muscle α-actin stress fibers and recruitment of proteins to cell-matrix adhesions. Old VSM cells presented an increase in integrin adhesion to the matrix and smooth muscle γ-actin fibers that was associated with increased cell stiffness. In conclusion, our results indicate that VSM contractile function declined with age in SFA. The decrement in contractile function was mediated in part by RhoA/ROCK signaling. Upregulation of pROCK in old VSM cells was not able to rescue contractility in old SFA. Collectively, these results indicate that changes at the VSM cell level play a central role in the reduced contractile function of aged SFA.

Restoring proper eNOS activity is key to ameliorating or preventing cardiovascular complications of diabetes. Continued investigation is needed to uncover new means for maintaining endothelial nitric oxide bioavailability.

Mutations in ACTA2, encoding smooth muscle α-actin, are a frequent cause of heritable thoracic aortic aneurysm and dissections. These mutations are associated with impaired vascular smooth muscle cell function, which leads to decreased ability of the cell to sense matrix-mediated mechanical stimuli. This study investigates how loss of smooth muscle α-actin affects cytoskeletal tension development and cell adhesion using smooth muscle cells explanted from aorta of mice lacking smooth muscle α-actin. We tested the hypothesis that reduced vascular smooth muscle contractility due to a loss of smooth muscle α-actin decreases cellular mechanosensing by dysregulating cell adhesion to the matrix. Assessment of functional mechanical properties of the aorta by stress relaxation measurements in thoracic aortic rings suggested two functional regimes for Acta2−/− mice. Lower stress relaxation was recorded in aortic rings from Acta2−/− mice at tensions below 10 mN compared with wild type, likely driven by cytoskeletal-dependent contractility. However, no differences were recorded between the two groups above the 10 mN threshold, since at higher tension the matrix-dependent contractility may be predominant. In addition, our results showed that at any given level of stretch, transmural pressure is lower in aortic rings from Acta2−/− mice than wild type mice. In addition, a three-dimensional collagen matrix contractility assay showed that collagen pellets containing Acta2−/− smooth muscle cells contracted less than the pellets containing the wild type cells. Moreover, second harmonic generation non-linear microscopy revealed that Acta2−/− cells locally remodeled the collagen matrix fibers to a lesser extent than wild type cells. Quantification of protein fluorescence measurements in cells also showed that in absence of smooth muscle α-actin, there is a compensatory increase in smooth muscle γ-actin. Moreover, specific integrin recruitment at cell–matrix adhesions was reduced in Acta2−/− cells. Thus, our findings suggest that Acta2−/− cells are unable to generate external forces to remodel the matrix due to reduced contractility and interaction with the matrix. Impact statement Thoracic aneurysm formation is characterized by progressive enlargement of the ascending aorta, which predisposes the aorta to acute aortic dissection that can lead to sudden death. SMCs in the aorta play an integral role in regulating vessel wall contractility and matrix deposition in the medial layer. Recent studies show that mutations in genes associated with actomyosin apparatus reduce SMC contractility, increasing susceptibility to TAAD. Single-cell experiments enable discrete measurements of transient microscopic events that may be masked by a macroscopic average tissue behavior. Biophysical methods combined with microscopy techniques aid in understanding the specific roles of adhesion and cytoskeletal proteins in regulating SMC mechanosensing when SMα-actin is disrupted. Our findings suggest that Acta2− /− cells have increased SMγ-actin and decreased integrin recruitment at cell–matrix adhesion, hence a synthetic phenotype with reduced cellular mechanosensing.