Background-Kinins are modulators of cardiovascular function. After ischemic injury, enhanced kinin generation may contribute in processes responsible for tissue healing. Methods and Results-Using pharmacological and genetic approaches, we investigated the role of kinin B 1 receptor in reparative angiogenesis in a murine model of limb ischemia. The effect of B 1 pharmacological manipulation on human endothelial cell proliferation and apoptosis was also studied in vitro. Abrogation of B 1 signaling dramatically inhibited the native angiogenic response to ischemia, severely compromising blood perfusion recovery. Outcome was especially impaired in B 1 knockouts that showed a very high incidence of limb necrosis, eventually leading to spontaneous auto-amputation. Conversely, local delivery of a long-acting B 1 receptor agonist enhanced collateral vascular growth in ischemic skeletal muscle, accelerated the rate of perfusion recovery, and improved limb salvage. In vitro, B 1 activation stimulated endothelial cell proliferation and survival, whereas B 1 antagonism induced apoptosis. Conclusions-Our results indicate that the B 1 plays an essential role in the host defense response to ischemic injury. B 1 signaling potentiation might be envisaged as a utilitarian target for the treatment of ischemic vascular disease.

Kinins play a central role in the modulation of cardiovascular function and in the pathophysiology of inflammation. These peptides mediate their effects by binding to two specific G-protein coupled receptors named B1 and B2. To evaluate the full functional relevance of the kallikrein-kinin system, we generated mice lacking both kinin receptors (B1B2-/-). Because of the close chromosomal position of both kinin receptor genes, B1B2-/- mice could not be obtained by simple breeding of the single knockout lines. Therefore, we inactivated the B1 receptor gene by homologous recombination in embryonic stem cells derived from B2-deficient animals. The B1B2-/- mice exhibited undetectable levels of mRNAs for both receptors and a lack of response to bradykinin (B2 agonist) and des-Arg9-bradykinin (B1 agonist), as attested by contractility studies with isolated smooth muscle tissues. B1B2-/- mice are healthy and fertile, and no sign of cardiac abnormality was detected. They are normotensive but exhibit a lower heart rate than controls. Furthermore, kinin receptor deficiency affects the pathogenesis of endotoxin-induced hypotension. While blood pressure decreased markedly in wild-type mice and B2-/- and moderately in B1-/- mice after bacterial lipopolysaccharide (LPS) injection, blood pressure remained unchanged in B1B2-/- mice. These results clearly demonstrate a pivotal role of kinins and their receptors in hypotension induced by endotoxemia in mice.

The kinin B1 receptor is an inducible receptor not normally expressed but induced by inflammatory stimuli and plays a major role in neutrophil recruitment, particularly in response to the cytokine interleukin-1β (IL-1β). However, the exact mechanism involved in this response is unclear. The aim of this study was to dissect the molecular mechanism involved, in particular to determine whether specific ELR-CXCL chemokines (specific neutrophil chemoattractants) played a role. Using intravital microscopy, we demonstrated that IL-1 β induced leukocyte rolling, adherence and emigration in mesenteric venules of wild type (WT) mice, associated with an increase of B1 receptor mRNA expression, was substantially attenuated (>80%) in B1 receptor knockout mice (B1KO). This effect in B1KO mice was correlated with a selective down regulation of IL-1β-induced CXCL5 mRNA and protein expression compared to WT mice. Furthermore a selective neutralizing CXCL5 antibody caused profound suppression of leukocyte emigration in IL-1β treated WT mice. Finally, treatment of human endothelial cells with IL-1β enhanced mRNA expression of B1 receptor and the human CXCL5 homologues (hCXCL5 and hCXCL6). This response was suppressed by ~50% when cells were pretreated with the B1 receptor antagonist des-Arg9[Leu]8BK whilst treatment with des-Arg9-BK, the B1 receptor agonist, caused a concentration-dependent increase in hCXCL5 and hCXCL6 mRNA expression. This study unveils a pro-inflammatory pathway centred on kinin B1 receptor activation of CXCL5 leading to leukocyte trafficking, and highlights the B1 receptor as a potential target in the therapeutics of inflammatory disease.

Abstract. A sexual dimorphism in hypertension has been observed in both human and laboratory animal studies. The mechanisms by which male sex hormones regulate cardiovascular homeostasis are still not yet fully understood and represent the subject of this study. The possible involvement of androgen receptors in the development of hypertension and end-organ damage in transgenic rats harboring the mouse Ren-2 renin gene [TGR(mREN2)27] was studied. Male TGR(mREN2)27 rats were treated with the androgen receptor antagonist Flutamide starting at 4 wk of age. Also, an androgen receptor mutation (testicular feminization mutation [tfm]) was introduced in these rats by crossbreeding male TGR(m-REN2)27 rats with tfm rats. The resulting offspring male rats that contain the tfm mutation are insensitive to androgens.Flutamide treatment or tfm mutation produced a significant attenuation of the development of hypertension. Besides a reduction in cardiac hypertrophy, urinary albumin excretion was blunted and no histologic characteristics of end-organ damage were observed in the kidney after Flutamide treatment. Testosterone levels increased 15-fold after Flutamide treatment and 2.7-fold by the tfm mutation. Also, plasma estrogens and luteinizing and follicle-stimulating hormones were significantly increased. Plasma renin concentrations and activity but not plasma angiotensinogen were reduced. Our results indicate that androgens contribute not only to the development of hypertension, but even more importantly to end-organ damage in TGR(mREN2)27 rats.Men are predisposed to hypertension and cardiovascular diseases more than age-matched, premenopausal women (1). A sexual dimorphism in hypertension has been observed both in human and laboratory animal studies (2). The mechanisms responsible for the gender differences in BP control are not yet clear and continue to be subject of active investigation (3). Evidence is accumulating that androgens may play an important role in gender-associated differences in BP regulation. Several studies have indicated that androgens may mediate hypertension and renal injury (4 -8). However, the mechanisms by which male sex hormones regulate cardiovascular homeostasis are not yet fully understood (3) and represent the subject of this study. There are indications for an interrelation between androgens and the renin-angiotensin system (RAS) (3); therefore, we studied the consequences of androgen receptor blockade on the development of malignant hypertension in transgenic TGR(mREN2)27 rats with overactive RAS (9,10). Androgen receptor inhibition was achieved by treatment with the antagonist Flutamide or by introducing a testicular feminization mutation (tfm) mutation, and BP as well as end-organ damage and RAS and pituitary-gonadal hormones were evaluated. Materials and Methods Rat StrainsMale transgenic heterozygous rats [TGR(mREN2)27] (n ϭ 24 rats) were obtained from the animal facilities of the Max-Delbrück-Center for Molecular Medicine, Berlin, Germany. Female Long-Evans rats carrying the X-linked rece...