Molecular Motor Contractıle Mechanısm of Retınal Perıcytes

Abstract

Although it is known that capillary pericytes function in the regulation of capillary tone and blood flow, the contractile proteins involved in these processes are unidentified. It is likely that α-SMA functions as a part of contractile apparatus in pericytes as it does in smooth muscle cells. Therefore, we examined the expression of α-SMA along the mouse retinal microvascular network together with the expression of Myh11 protein, which we identified as the potential myosin heavy chain isoform candidate based on the single cell transcriptomic data in the literature. We found that α-SMA is expressed by capillary pericytes, but depolymerization must be prevented for its demonstration in downstream segments, in contrast to Myh11, which highly colocalizes with α-SMA but is present throughout the microvascular tree. Immunofluorescent signals delineated close association of α-SMA and Myh11, which was also confirmed by high FRET efficiency, oriented as thin strands reminiscent of stress-fibers, conforming to a contractile function. The functional correspondence of this spatial association was evaluated by inhibition of actomyosin cross-bridge cycling with blebbistatin, which prevented noradrenaline-induced vasoconstriction. Alongside, a supportive mechanism in cellular contraction, actin polymerization, was assessed using markers identifying filamentous and globular forms of actin. After intravitreal noradrenaline application, actin polymerization was pronounced in downstream capillary pericytes, suggesting a need for actin polymerization for contractile force generation because they express α-SMA in low quantities. In conclusion, pericytes contract using actomyosin cross-bridge cycling, which is supplemented by actin polymerization in downstream pericytes.