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Tissue kallikrein is essential for invasive capacity of circulating proangiogenic cells.

ABSTRACT: Homing of proangiogenic cells (PACs) is guided by chemoattractants and requires proteases to disrupt the extracellular matrix. The possibility that PAC recruitment involves an interaction between proteases and chemotactic factor receptors remains largely unexplored.To determine the role of human tissue kallikrein (hK1) in PAC invasion and its dependency on kinin receptor signaling.Human mononuclear cells (MNCs) and culture-selected PACs express and release mature hK1 protein. HK1 gene (KLK1) silencing reduced PACs migratory, invasive, and proangiogenic activities. KLK1-knockout mouse bone marrow-derived MNCs showed similar impairments and were unable to support reparative angiogenesis in a mouse model of peripheral ischemia. Conversely, adenovirus-mediated KLK1 (Ad.KLK1) gene transfer enhanced PAC-associated functions, whereas the catalytically inactive variant R53H-KLK1 was ineffective. HK1-induced effects are mediated by a kinin B(2) receptor (B(2)R)-dependent mechanism involving inducible nitric oxide synthase and metalloproteinase-2 (MMP2). Lower hK1 protein levels were observed in PACs from type 2 diabetic (T2D) patients, whereas KLK1 mRNA levels were similar to those of healthy subjects, suggesting a post-transcriptional defect. Furthermore, B(2)R is normally expressed on T2D-PACs but remains uncoupled from downstream signaling. Importantly, whereas Ad.KLK1 alone could not restore T2D-PAC invasion capacity, combined KLK1 and B(2)R expression rescued the diabetic phenotype.This study reveals new interactive components of the PACs invasive machinery, acting via protease- and kinin receptor-dependent mechanisms.

SUBMITTER: Spinetti G

PROVIDER: S-EPMC3596779 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Tissue kallikrein is essential for invasive capacity of circulating proangiogenic cells.

Spinetti Gaia G Fortunato Orazio O Cordella Daniela D Portararo Paola P Kränkel Nicolle N Katare Rajesh R Sala-Newby Graciela B GB Richer Christine C Vincent Marie-Pascale MP Alhenc-Gelas Francois F Tonolo Giancarlo G Cherchi Sara S Emanueli Costanza C Madeddu Paolo P

Circulation research 20101216 3

<h4>Rationale</h4>Homing of proangiogenic cells (PACs) is guided by chemoattractants and requires proteases to disrupt the extracellular matrix. The possibility that PAC recruitment involves an interaction between proteases and chemotactic factor receptors remains largely unexplored.<h4>Objective</h4>To determine the role of human tissue kallikrein (hK1) in PAC invasion and its dependency on kinin receptor signaling.<h4>Methods and results</h4>Human mononuclear cells (MNCs) and culture-selected ...[more]

PMID: 21164105

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Project description:RATIONALE:Circulating proangiogenic cells (PACs) support postischemic neovascularization. Cardiovascular disease and diabetes mellitus impair PAC regenerative capacities via molecular mechanisms that are not fully known. We hypothesize a role for microRNAs (miRs). Circulating miRs are currently investigated as potential diagnostic and prognostic biomarkers. OBJECTIVE:The objectives were the following: (1) to profile miR expression in PACs from critical limb ischemia (CLI) patients; (2) to demonstrate that miR-15a and miR-16 regulate PAC functions; and (3) to characterize circulating miR-15a and miR-16 and to investigate their potential biomarker value. METHODS AND RESULTS:Twenty-eight miRs potentially able to modulate angiogenesis were measured in PACs from CLI patients with and without diabetes mellitus and controls. miR-15a and miR-16 were further analyzed. CLI-PACs expressed higher level of mature miR-15a and miR-16 and of the primary transcript pri-miR-15a/16-1. miR-15a/16 overexpression impaired healthy PAC survival and migration. Conversely, miR-15a/16 inhibition improved CLI-PAC-defective migration. Vascular endothelial growth factor-A and AKT-3 were validated as direct targets of the 2 miRs, and their protein levels were reduced in miR-15a/16-overexpressing healthy PACs and in CLI-PACs. Transplantation of healthy PACs ex vivo-engineered with anti-miR-15a/16 improved postischemic blood flow recovery and muscular arteriole density in immunodeficient mice. miR-15a and miR-16 were present in human blood, including conjugated to argonaute-2 and in exosomes. Both miRs were increased in the serum of CLI patients and positively correlated with amputation after restenosis at 12 months postrevascularization of CLI type 2 diabetes mellitus patients. Serum miR-15a additionally correlated with restenosis at follow-up. CONCLUSIONS:Ex vivo miR-15a/16 inhibition enhances PAC therapeutic potential, and circulating miR-15a and miR-16 deserves further investigation as a prognostic biomarker in CLI patients undergoing revascularization.

Project description:The function of human circulating proangiogenic cells (PACs) has been described extensively. However, little focus has been placed on understanding how these cells differ in their functions in the presence of microenvironments mimicking vascular inflammation. We hypothesized that exposure to proinflammatory cytokines or the oxLDL, an autoantigen abundant in advanced atherosclerotic plaques, converts PACs into immune-modulating/proinflammatory cells. Hence, we examined the effect of oxLDL and inflammatory stimuli on their phenotype by use of a functional genomics model based on secretome and whole genome transcriptome profiling. PACs obtained from culturing a PBMC fraction in angiogenic medium were primed with DC differentiation cytokines for 3 days and then exposed to proinflammatory cytokines or oxLDL for 2 days. Under these conditions, PACs converted into APCs (APCcy and APCox, respectively), expressed maturation markers CD80 and CD83, and showed an up-regulation of CD86. APCcy and APCox induced a robust T cell BrdU incorporation. Despite a similar ability to induce lymphocyte proliferation, APCcy and APCox differed for the secretory pathway and mRNA expression. Analysis of the differentially expressed genes identified 4 gene "clusters", showing reciprocal modulation in APCcy vs. APCox justifying, according to functional genomics analyses, a different putative function of the cells in antigen processing. Together, these data show that treatment with inflammatory cytokines or oxLDL converts human PAC phenotype and function into that of APCs with similar lymphocyte-activating ability but distinct maturation degree and paracrine functions. PBMC-derived PACs were committed with GM-CSF and IL-4 to a dendritic phenotype, and then exposed to proinflammatory cytokines (IL-1β and TNF-α) or to the oxidized form of LDL (oxLDL). Functional assays and whole genome gene expression profiling were performed both on PACs (n=6) and or resulting cytokine- (APCcy, n=6) and oxLDL-induced APCs (APCox, n=6). RNA was reverse transcribed, labeled, and linearly amplified, and then hybridized to HumanHT-12 v.4 Expression BeadChip microarrays. To estimate technical variability, ≈20% of the samples (n=4) were hybridized in duplicate.

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Tissue kallikrein is essential for invasive capacity of circulating proangiogenic cells.

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Tissue kallikrein is essential for invasive capacity of circulating proangiogenic cells.

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