Abstract
Background: Circulating proangiogenic cells (PACs) express the serine protease tissue kallikrein (K1), which favours invasion through metalloproteinases activation. Aim: To determine whether inherited or disease-induced K1 deficiency alters PACs migration and proangiogenic capacity, and whether K1-deficient PACs transduction with wild type or polymorphic variant (R53H) K1 can rescue functional defects. Methods & Results: Wild type (WT, n=8) or K1-knockout mice (K1KO, n=7) lineage negative (Lin-) bone marrow mononuclear cells (BMMNC) were used in migration assays or differentiated into PACs. K1KO Lin-BMMNC showed defective migration to SDF-1 α compared to WT (fold increase vs vehicle 2.2, p=N.S and 5.8, p<0.05 vs vehicle and K1KO, respectively, n=3). Similarly impaired was K1KO PACs migration (n=3, p<0.05 vs WT). Further, K1KO PACs were unable to promote endothelial cell (EC) networking in vitro (p<0.05). Adenovirus-mediated K1 transduction rescued both defects in K1KO PACs (n=3, p<0.05 vs Ad.Null). Next, we studied type 2 diabetic patients and age-matched healthy controls circulating PACs (n<6 per group). Diabetic PACs showed reduced migration and proangiogenic potential (−1.8 and 2.0 fold vs controls, p<0.05) and lower K1 protein (−56.6 %, p<0.05), but normal K1 mRNA abundance, pointing at a post-transcriptional defect. K1 overexpression increased healthy PACs migration and EC networking (p<0.01 vs Ad.Null), but could not revert diabetic PACs liabilities (p±N.S. vs Ad.Null). We then verified if K1 increases PACs invasive capacity. K1-PACs invaded matrix more efficiently (2.5-fold increase, p<0.01 vs Null-PACs). In contrast, the polymorphic variant R53H, which encodes for an enzymatic defective K1 form, did not improve invasion. Moreover, K1-PACs failed to invade the matrix when regular collagen was substituted with glycated one. Conclusions: Inherited or diabetes-induced K1 deficit hampers PACs invasive and proangiogenic capacities, with K1 gene therapy being able to restore those functions except in diabetes. Diabetes could interfere with K1 enzymatic activity and make extracellular matrix impenetrable to PACs. Alternatively, additional migratory machinery deficit may overwhelm benefits of K1 overexpression.
Published Version
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