Participation of kinins in the captopril-induced inhibition of intimal hyperplasia caused by interruption of carotid blood flow in the mouse

1. In the rat balloon injury model, angiotensin-converting enzyme (ACE) inhibitors prevent vascular remodelling by inhibiting angiotensin II generation and kinin breakdown. We investigated if ACE inhibition also prevents the structural vascular responses to disruption of carotid artery blood flow and if kinin potentiation plays a role in such a protection. 2. Morphometric analysis of the structural alterations caused by ligation of the left carotid artery was performed 14 days after surgery in J129Sv wild-type mice (B(2)(+/+)) drinking normal tap water or water containing captopril (120 mg kg(-1) per day). In addition, the effect of captopril on vascular remodelling was tested in B(2)(+/+) given the bradykinin (BK) B(1) receptor antagonist des-Arg(9)-[Leu(8)]-BK (DALBK, 50 nmol kg(-1) per day, intraperitoneally) or the BK B(2) receptor antagonist D-Arg, [Hyp(3),Thi(5)D-Tic(7),Oic(8)]-BK (icatibant, 1 micromol kg(-1) per day, intraperitoneally), and in B(2) receptor gene knockout mice (B(2)(-/-)). 3. Interruption of blood flow resulted in carotid artery intimal hyperplasia and media thickening in untreated B(2)(+/+), these responses being partially suppressed by captopril. The inhibition of intimal thickening exerted by captopril was reduced in B(2)(+/+) given DALBK or icatibant (P<0.05 for both comparisons) as well as in B(2)(-/-) (P<0.05). Neither antagonism of kinin receptors nor disruption of the B(2) receptor gene altered the suppressive effect of captopril on media thickening. The protection of vascular wall structure was independent of the reduction in blood pressure by captopril. 4. These results demonstrate that kinins participate in the inhibitory effect of captopril on intimal hyperplasia via B(1) and B(2) receptor signalling. Our findings may have important implications in treating vascular remodelling evoked by altered shear stress conditions.     

Nonviral and viral gene transfer to the kidney in the context of transplantation

BACKGROUND/AIMS: Local modulation of the immune response through genetic manipulation of the graft is an attractive novel approach to overcome the toxicity of immunosuppressive therapy to prevent acute graft rejection. We have previously reported that the cationic polymer polyethylenimine 25k (PEI 25k) transduced reporter genes when injected into the renal artery, but with a low transfection efficiency. Here we compare the risk/benefit profiles of such a nonviral versus a viral technique of gene transfer to the kidney in the context of renal transplantation. METHODS: Donor kidneys from Lewis rats were perfused in a closed circuit with an artificial cell-free medium containing PEI 25k complexed to an expression vector coding for the beta-galactosidase (beta-gal) gene and subsequently transplanted in a syngeneic animal. In a second set of experiments, donor kidneys were injected or perfused with a replication-deficient adenovirus encoding the beta-gal gene (AdCMV. beta-gal; 1 x 10(9) plaque-forming units) before transplantation. RESULTS: Perfusion of the kidney with PEI 25k/DNA complexes resulted in large areas of hypoperfusion characterized by injured glomeruli and tubuli, capillary thrombosis and accumulation of C3 in glomerular capillaries. Reperfusion of the kidney was achieved by lowering the PEI 25k/DNA ratio, but no detectable transfection was observed. In animals receiving adenovirus, the beta-gal activity increased with time and was localized mainly in proximal and distal tubular cells, as documented by beta-gal histochemistry and in situ hybridization. A significantly increased expression of beta-gal was achieved by perfusion of the kidney with AdCMV.beta-gal before transplantation, beta-gal staining mainly localizing in proximal and distal tubular cells. CONCLUSIONS: Unlike nonviral methods of gene delivery, adenovirus-mediated gene transfer to the kidney offers exciting perspectives for the development of molecular medicine in the field of organ transplantation.     

RGDS peptide induces caspase 8 and caspase 9 activation in human endothelial cells

Peptides containing the Arg-Gly-Asp (RGD) motif inhibit cell adhesion and exhibit a variety of other biologic effects including anticoagulant and antimetastatic activities. The aim of the present study was to examine the anchorage-independent effects of an RGD-containing peptide, Arg-Gly-Asp-Ser (RGDS), on human umbilical vein endothelial cells (HUVECs). Assays were performed on HUVECs seeded onto collagen IV; under these experimental conditions RGDS did not exert antiadhesive effects but significantly reduced FGF-2-dependent chemotaxis after 4 hours of treatment and reduced proliferation after 24 hours of treatment. Experiments carried out with caspase-specific inhibitors indicated that the observed antichemotactic effects required caspase 8 and caspase 9 activation. RGDS activated both caspase 8 and caspase 9 after 4 hours of treatment and caspase 3 after 24 hours of treatment, and markedly enhanced HUVEC apoptosis by transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)/Hoechst staining and fluorescence-activated cell sorting (FACS) analysis. Finally, confocal microscopy showed that RGDS localizes in the cytoplasm of live HUVECs within 4 hours and in vitro experiments showed that RGDS directly interacts with recombinant caspases 8 and 9 in a specific way. In summary, these results indicate that RGDS directly binds and activates caspases 8 and 9, inhibits chemotaxis, and induces apoptosis of HUVECs with a mechanism independent from its antiadhesive effect.     

Differences in microparticle release in patients with acute coronary syndrome and stable angina

Background:?Microparticles (MP) are vesicles released from activated or apoptotic cells. Endothelial MP (EMP) are derived from injured endothelium, platelet MP (PMP) from activated platelets, and Annexin V positive MP (AMP) from apoptotic endothelial cells. The aim was to assess the release of MP and its association with inflammation and atherosclerotic burden. Methods and Results:?AMP, EMP and PMP were measured on admission (Day 0) in 33 patients with stable angina (SA) and 43 patients with acute coronary syndrome (ACS) undergoing percutaneous coronary interventions (PCI). In SA, peripheral artery disease (PAD) was assessed by ultrasound examination. In 30 of the 76 patients (20 ACS and 10 SA), MP, high-sensitivity-C-reactive protein (hs-CRP), and troponin T (TnT) levels were also assessed 24h (Day 1) and 48h (Day 2) after PCI. AMP, EMP, and PMP were higher in ACS than in SA (all P<0.01). In the SA group, AMP, PMP, and EMP were similar in patients with or without PAD. In the ACS group, AMP increased until Day 2 (P=0.001), while EMP and PMP peaked on Day 1 (P<0.01) then decreased to baseline values. Day 2 AMP correlated with Day 2 TnT levels (r=0.43, P=0.01) while Day 1 EMP and PMP correlated with Day 1 hs-CRP (r=0.37, P=0.04 and r=0.33, P=0.05; respectively). Conclusions:?Higher MP levels were observed in ACS than in SA. Atherosclerotic burden did not affect MP levels in stable patients. (Circ J?2012; 76: 2174-2182).     

NO points to epigenetics in vascular development

Our understanding of epigenetic mechanisms important for embryonic vascular development and cardiovascular differentiation is still in its infancy. Although molecular analyses, including massive genome sequencing and/or in vitro/in vivo targeting of specific gene sets, has led to the identification of multiple factors involved in stemness maintenance or in the early processes of embryonic layers specification, very little is known about the epigenetic commitment to cardiovascular lineages. The object of this review will be to outline the state of the art in this field and trace the perspective therapeutic consequences of studies aimed at elucidating fundamental epigenetic networks. Special attention will be paid to the emerging role of nitric oxide in this field.