Capillary Pericytes Express Α-Smooth Muscle Actin, Which Requires Prevention of Filamentous-Actin Depolymerization For Detection
Di Polo, Adriana
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Recent evidence suggests that capillary pericytes are contractile and play a crucial role in the regulation of microcirculation. However, failure to detect components of the contractile apparatus in capillary pericytes, most notably α-smooth muscle actin (α-SMA), has questioned these findings. Using strategies that allow rapid filamentous-actin (F-actin) fixation (i.e. snap freeze fixation with methanol at −20°C) or prevent F-actin depolymerization (i.e. with F-actin stabilizing agents), we demonstrate that pericytes on mouse retinal capillaries, including those in intermediate and deeper plexus, express α-SMA. Junctional pericytes were more frequently α-SMA-positive relative to pericytes on linear capillary segments. Intravitreal administration of short interfering RNA (α-SMA-siRNA) suppressed α-SMA expression preferentially in high order branch capillary pericytes, confirming the existence of a smaller pool of α-SMA in distal capillary pericytes that is quickly lost by depolymerization. We conclude that capillary pericytes do express α-SMA, which rapidly depolymerizes during tissue fixation thus evading detection by immunolabeling., Blood vessels in animals’ bodies are highly organized. The large blood vessels from the heart branch to smaller vessels that are spread throughout the tissues. The smallest vessels, the capillaries, allow oxygen and nutrients to pass from the blood to nearby cells in tissues. Some capillaries, including those at the back of the eye (in the retina) and those in the brain, change their diameter in response to activity in the nervous system. This allows more or less oxygen and nutrients to be delivered to match these tissues’ demands. However, unlike for larger blood vessels, how capillaries constrict or dilate is debated., While large vessels are encircled by smooth muscle cells, capillaries are instead surrounded by muscle-like cells called pericytes, and some scientists have suggested that it is these cells that contract to narrow the diameter of a capillary or relax to widen it. However, other researchers have questioned this explanation. This is mostly because several laboratories could not detect the proteins that would be needed for contraction within these pericytes – the most notable of which is a protein called α-smooth muscle actin (or α-SMA for short)., Alarcon-Martinez, Yilmaz-Ozcan et al. hypothesized that the way samples are usually prepared for analysis was causing the α-SMA to be degraded before it could be detected. To test this hypothesis, they used different methods to fix and preserve capillaries and pericytes in samples taken from the retinas of mice. When the tissue samples were immediately frozen with ice-cold methanol instead of a more standard formaldehyde solution, α-SMA could be detected at much higher levels in the capillary pericytes. Treating samples with a toxin called phalloidin, which stabilizes filaments of actin, also made α-SMA more readily visible. When α-SMA was experimentally depleted from the mouse retinas, the capillary pericytes were more affected than the larger blood vessels. This finding supports the idea that the pericytes contain, and rely upon, only a small amount of α-SMA., Finding α-SMA in capillary pericytes may explain how these small blood vessels can change their diameter. Future experiments will clarify how these pericytes regulate blood flow at the level of individual capillaries, and may give insights into conditions such as stroke, which is caused by reduced blood flow to the brain.