Description: Objective— Kindlin-3 is a critical supporter of integrin function in platelets. Lack of expression of kindlin-3 protein in patients impairs integrin αIIbβ3–mediated platelet aggregation. Although kindlin-3 has been categorized as an integrin-binding partner, the functional significance of the direct interaction of kindlin-3 with integrin αIIbβ3 in platelets has not been established. Here, we evaluated the significance of the binding of kindlin-3 to integrin αIIbβ3 in platelets in supporting integrin αIIbβ3–mediated platelet functions. Approach and Results— We generated a strain of kindlin-3 knockin (K3KI) mice that express a kindlin-3 mutant that carries an integrin-interaction defective substitution. K3KI mice could survive normally and express integrin αIIbβ3 on platelets similar to their wild-type counterparts. Functional analysis revealed that K3KI mice exhibited defective platelet function, including impaired integrin αIIbβ3 activation, suppressed platelet spreading and platelet aggregation, prolonged tail bleeding time, and absence of platelet-mediated clot retraction. In addition, whole blood drawn from K3KI mice showed resistance to in vitro thrombus formation and, as a consequence, K3KI mice were protected from in vivo arterial thrombosis. Conclusions— These observations demonstrate that the direct binding of kindlin-3 to integrin αIIbβ3 is involved in supporting integrin αIIbβ3 activation and integrin αIIbβ3-dependent responses of platelets and consequently contributes significantly to arterial thrombus formation.

Description: Objective— To investigate the novel function of ASK1-interacting protein-1 (AIP1) in vascular endothelial cell growth factor receptor (VEGFR)-3 signaling, and VEGFR-3–dependent angiogenesis and lymphangiogenesis. Approach and Results— AIP1, a signaling scaffold protein, is highly expressed in the vascular endothelium. We have previously reported that AIP1 functions as an endogenous inhibitor in pathological angiogenesis by blocking VEGFR-2 activity. Surprisingly, here we observe that mice with a global deletion of AIP1-knockout mice (AIP1-KO) exhibit reduced retinal angiogenesis with less sprouting and fewer branches. Vascular endothelial cell (but not neuronal)–specific deletion of AIP1 causes similar defects in retinal angiogenesis. The reduced retinal angiogenesis correlates with reduced expression in VEGFR-3 despite increased VEGFR-2 levels in AIP1-KO retinas. Consistent with the reduced expression of VEGFR-3, AIP1-KO show delayed developmental lymphangiogenesis in neonatal skin and mesentery, and mount weaker VEGF-C–induced cornea lymphangiogenesis. In vitro, human lymphatic endothelial cells with AIP1 small interfering RNA knockdown, retinal endothelial cells, and lymphatic endothelial cells isolated from AIP1-KO all show attenuated VEGF-C–induced VEGFR-3 signaling. Mechanistically, we demonstrate that AIP1 via vegfr-3 –specific miR-1236 increases VEGFR-3 protein expression and that, by directly binding to VEGFR-3, it enhances VEGFR-3 endocytosis and stability. Conclusion— Our in vivo and in vitro results provide the first insight into the mechanism by which AIP1 mediates VEGFR-3–dependent angiogenic and lymphangiogenic signaling.

Description: Rationale: PKP2 , encoding plakophilin 2 (PKP2), is the most common causal gene for arrhythmogenic cardiomyopathy. Objective: To characterize miRNA expression profile in PKP2-deficient cells. Methods and Results: Control and PKP2-knockdown HL-1 (HL-1 Pkp2-shRNA ) cells were screened for 750 miRNAs using low-density microfluidic panels. Fifty-nine miRNAs were differentially expressed. MiR-184 was the most downregulated miRNA. Expression of miR-184 in the heart and cardiac myocyte was developmentally downregulated and was low in mature myocytes. MicroRNA-184 was predominantly expressed in cardiac mesenchymal progenitor cells. Knockdown of Pkp2 in cardiac mesenchymal progenitor cells also reduced miR-184 levels. Expression of miR-184 was transcriptionally regulated by the E2F1 pathway, which was suppressed in PKP2-deficient cells. Activation of E2F1, on overexpression of its activator CCND1 (cyclin D1) or knockdown of its inhibitor retinoblastoma 1, partially rescued miR-184 levels. In addition, DNA methyltransferase-1 was recruited to the promoter region of miR-184, and the CpG sites at the upstream region of miR-184 were hypermethylated. Treatment with 5-aza-2'-deoxycytidine, a demethylation agent, and knockdown of DNA methyltransferase-1 partially rescued miR-184 level. Pathway analysis of paired miR-184:mRNA targets identified cell proliferation, differentiation, and death as the main affected biological processes. Knockdown of miR-184 in HL-1 cells and mesenchymal progenitor cells induced and, conversely, its overexpression attenuated adipogenesis. Conclusions: PKP2 deficiency leads to suppression of the E2F1 pathway and hypermethylation of the CpG sites at miR-184 promoter, resulting in downregulation of miR-184 levels. Suppression of miR-184 enhances and its activation attenuates adipogenesis in vitro. Thus, miR-184 contributes to the pathogenesis of adipogenesis in PKP2-deficient cells.