Background: Considerable evidence indicates a key role for primary cilia of mammalian cells in mechanochemical sensing. Dysfunctions of primary cilia have been linked to the pathogenesis of several human diseases. However, cilia-related research has been limited to a few cell and tissue types; to our knowledge, no literature exists on primary cilia in airway smooth muscle (ASM). The aim of this study was to characterize primary cilia in human ASM. Methods: Primary cilia of human bronchial smooth muscle cells (HBSMCs) were examined using immunofluorescence confocal microscopy, and scanning and transmission electron microscopy. HBSMC migration and injury repair were examined by scratch-wound and epidermal growth factor (EGF)-induced migration assays.

Although primary cilia are increasingly recognized to play sensory roles in several cellular systems, their role in vascular smooth muscle cells (VSMCs) has not been defined. We examined in situ position/orientation of primary cilia and ciliary proteins in VSMCs and tested the hypothesis that primary cilia of VSMCs exert sensory functions. By immunofluorescence and electron microscopic imaging, primary cilia of VSMCs were positioned with their long axis aligned at 58.3° angle in relation to the cross-sectional plane of the artery, projecting into the extracellular matrix (ECM). Polycystin-1, polycystin-2 and α3- and β1-integrins are present in cilia. In scratch wound experiments, the majority of cilia were repositioned to the cell-wound interface. Such repositioning was largely abolished by a β1-integrin blocker. Moreover, compared to non-ciliated/deciliated cells, ciliated VSMCs showed more efficient migration in wound repair. Lastly, when directly stimulated with collagen (an ECM component and cognate ligand for α3β1-integrins) or induced ciliary deflection, VSMCs responded with a rise in [Ca²⁺]_i that is dependent on the presence of cilia. Taken together, primary cilia of VSMCs are preferentially oriented, possess proteins critical for cell-ECM interaction and mechanosensing and respond to ECM protein and mechanical stimulations. These observations suggest a role for primary cilia in mechanochemical sensing in vasculature.

Vascular complications are the leading cause of morbidity and mortality in autosomal dominant polycystic kidney disease. Although evidence suggests an abnormal vascular reactivity, contractile function in Pkd mutant vessels has not been studied previously. Contractile response to phenylephrine (PE; 10 ؊10 to 10 ؊4 M), an ␣1-adrenergic receptor agonist, was examined.De-endothelialized Pkd2 ϩ/Ϫ aortic rings generated a higher maximum force (

The mechanisms underlying vascular complications in autosomal-dominant polycystic kidney disease (ADPKD) have not been fully elucidated. However, molecular components altered in Pkd mutant vascular smooth muscle cells (VSMCs) are gradually being identified. Pkd2+/− arterial smooth muscles show elevated levels of (1) phenylephrine (PE)-induced, Ca2+-independent vasocontraction and (2) smooth muscle α-actin (SMA) expression. As these two processes are heavily influenced by RhoA signaling and by cellular filamentous-to-globular (F/G)-actin dynamics, we examined PE-induced changes in RhoA activation and the F/G-SMA ratio in wild-type (wt) and Pkd2+/− VSMCs; we further tested the hypothesis that the abnormal response to PE and the resultant elevation in the F/G-SMA ratio contribute to the exuberant SMA expression in Pkd2+/− VSMCs. GTP-RhoA and F/G-SMA in mouse aortic media and primary cultured VSMCs were determined using RhoA activation and in vivo F-to-G-actin assays. Myocardin-related transcription factor-A (MRTF-A) (SMA transcription coactivator) was localized by immunofluorescence, nuclear MRTF-A quantified by western analysis using nuclear extracts and SMA expression by luciferase reporter assay. PE induced a >3-fold higher RhoA activation in Pkd2+/− than in wt VSMCs and higher levels of downstream p-LIMK and p-cofilin. Moreover, Pkd2+/− VSMCs showed a higher baseline and PE-induced F/G-SMA ratio. The F/G-SMA elevation enhanced nuclear translocation of MRTF-A, which upregulated SMA transcription. In summary, PE-induced RhoA hyperactivation and defects in F-to-G SMA balance likely have a role in the abnormal vasocontraction and SMA expression in Pkd2+/− arteries. These defects could potentially contribute to the genesis of vascular complications in ADPKD, thus providing new areas for further research and therapeutic targeting.