turbidimetry-on-human-washed-platelets-effect-pannexin1-inhibitor

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or ...

University of Geneva

Cannabinoid receptor CB(2) activation inhibits inflammatory proliferation and migration of vascular smooth muscle cells in vitro. The potential in vivo relevance of these findings is unclear. We performed carotid balloon distension injury in hypercholesterolemic apolipoprotein E knockout (ApoE(-/-)) mice receiving daily intraperitoneal injection of the CB(2) agonist JWH133 (5 mg/kg) or vehicle, with the first injection given 30 min before injury. Alternatively, we subjected CB(2)(-/-) and wild-type (WT) mice to balloon injury. We determined CB(2) mRNA and protein expression in dilated arteries of ApoE(-/-) mice. Neointima formation was assessed histologically. We used bone marrow-derived murine CB(2)(-/-) and WT macrophages to study adhesion to plastic, fibronectin, or collagen, and migration was assayed by modified Boyden chamber. Aortic smooth muscle cells were isolated to determine in vitro proliferation rates. We found increased vascular CB(2) expression in ApoE(-/-) mice in response to balloon injury. Seven to twenty-one days after dilatation, injured vessels of JWH133-treated mice had less intimal nuclei numbers as well as intimal and medial areas, associated with less staining for proliferating cells, smooth muscle cells, and macrophages. Complete endothelial repair was observed after 14 days in both JWH133- and vehicle-treated mice. CB(2) deficiency resulted in increased intima formation compared with WT, whereas JWH133 did not affect intimal formation in CB(2)(-/-) mice. Apoptosis rates assessed by in situ terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining 1 h postballooning were significantly higher in the CB(2) knockouts. In vitro, bone marrow-derived CB(2)(-/-) macrophages showed enhanced adherence and migration compared with WT cells and elevated mRNA levels of adhesion molecules, chemokine receptors CCR1 and 5, and chemokine CCL2. Proliferation rates were significantly increased in CB(2)(-/-) smooth muscle cells compared with WT. In conclusion, pharmacological activation or genetic deletion of CB(2) receptors modulate neointima formation via protective effects in macrophages and smooth muscle cells.

Cannabinoid receptor CB2 protects against balloon-induced neointima formation.

Endocannabinoids are endogenous bioactive lipids ubiquitously distributed in several tissues (e.g., brain, adipose tissue, liver, heart and arterial vessels), which play a crucial role in atherosclerosis. Endocannabinoids have been shown to promote cell homeostasis and modulate inflammatory bioactivities mainly via the binding to transmembrane receptors (called cannabinoid type 1 and cannabinoid type 2 receptors, respectively). Although other cannabinoid receptors have been recently identified and shown to play a crucial role in cardiovascular pathophysiology, so far, the pharmacological targeting of both cannabinoid type 1 and cannabinoid type 2 receptors has been described as a promising therapeutic target in atherogenesis and associated inflammatory processes. In particular, endocannabinoids have been shown to modulate the release and activation of matrix degrading enzymes (i.e., matrix metalloproteinases [MMPs]) increasing intraplaque vulnerability. In this article the authors describe the pivotal regulatory activity of the endocannabinoid system on gelatinase (MMP-2 and -9) bioactivity in the arterial wall physiology and pathophysiology.

Update on the endocannabinoid-mediated regulation of gelatinase release in arterial wall physiology and atherosclerotic pathophysiology.

Percutaneous transluminal angioplasty is frequently used in patients with severe arterial narrowing due to atherosclerosis. However, it induces severe arterial injury and an inflammatory response leading to restenosis. Here, we studied a potential activation of the endocannabinoid system and the effect of FA amide hydrolase (FAAH) deficiency, the major enzyme responsible for endocannabinoid anandamide degradation, in arterial injury. We performed carotid balloon injury in atherosclerosis-prone apoE knockout (apoE(-/-)) and apoE(-/-)FAAH(-/-) mice. Anandamide levels were systemically elevated in apoE(-/-) mice after balloon injury. ApoE(-/-)FAAH(-/-) mice had significantly higher baseline anandamide levels and enhanced neointima formation compared with apoE(-/-) controls. The latter effect was inhibited by treatment with CB1 antagonist AM281. Similarly, apoE(-/-) mice treated with AM281 had reduced neointimal areas, reduced lesional vascular smooth-muscle cell (SMC) content, and proliferating cell counts. The lesional macrophage content was unchanged. In vitro proliferation rates were significantly reduced in CB1(-/-) SMCs or when treating apoE(-/-) or apoE(-/-)FAAH(-/-) SMCs with AM281. Macrophage in vitro adhesion and migration were marginally affected by CB1 deficiency. Reendothelialization was not inhibited by treatment with AM281. In conclusion, endogenous CB1 activation contributes to vascular SMC proliferation and neointima formation in response to arterial injury.

Endogenous cannabinoid receptor CB1 activation promotes vascular smooth-muscle cell proliferation and neointima formation.

Connexins (Cxs) form a family of transmembrane proteins comprising 21 members in humans. Cxs differ in their expression patterns, biophysical properties and ability to combine into homomeric or heteromeric gap junction channels between neighbouring cells. The permeation of ions and small metabolites through gap junction channels or hemichannels confers a crucial role to these proteins in intercellular communication and in maintaining tissue homeostasis. Among others, Cx37, Cx40, Cx43, Cx45 and Cx47 are found in heart, blood and lymphatic vessels. Mutations or polymorphisms in the genes coding for these Cxs have not only been implicated in cardiovascular pathologies but also in a variety of other disorders. While mutations in Cx43 are mostly linked to oculodentodigital dysplasia, Cx47 mutations are associated with Pelizaeus-Merzbacher-like disease and lymphoedema. Cx40 mutations are principally linked to atrial fibrillation. Mutations in Cx37 have not yet been described, but polymorphisms in the Cx37 gene have been implicated in the development of arterial disease. This review addresses current knowledge on gene mutations in cardiovascular Cxs systematically and links them to alterations in channel properties and disease.

Mutations in cardiovascular connexin genes.

Obesity, and especially excessive visceral adipose tissue accumulation, is considered as a low-grade inflammatory state that is responsible for adipocyte dysfunction and associated metabolic disorders. Adipose tissue displays endocrine functions by releasing pro- or anti-inflammatory bioactive molecules named adipokines. An altered expression of these molecules, provoked by obesity or adipocyte dysregulation, contributes to major metabolic diseases such as insulin resistance and type 2 diabetes mellitus that are important risk factors for cardiovascular disease. However, obesity is also characterised by the expansion of perivascular adipose tissue that acts locally via diffusion of adipokines into the vascular wall. Local inflammation within blood vessels induced by adipokines contributes to the onset of endothelial dysfunction, atherosclerosis and thrombosis, but also to vascular remodelling and hypertension. A fast expansion of obesity is expected in the near future, which will rapidly increase the incidence of these cardiovascular diseases. The focus of this review is to summarise the link between metabolic and cardiovascular disease and discuss current treatment approaches, limitations and future perspectives for more targeted therapies.

Adipokines at the crossroad between obesity and cardiovascular disease.

Pannexin1 (Panx1), a membrane channel-forming protein permitting the passage of small-sized molecules, such as ATP, is expressed in human platelets. Recently, we showed that inhibiting Panx1 affects collagen-induced platelet aggregation but not aggregation triggered by other agonists. We also found that a single nucleotide polymorphism (SNP; rs1138800) in the Panx1 gene encoded for a gain-of-function channel (Panx1-400C) and was associated with enhanced collagen-induced platelet reactivity. Here, we assessed the association of this SNP with platelet reactivity in a cohort of 758 stable cardiovascular patients from the ADRIE study treated with aspirin and/or clopidogrel. We found that presence of the Panx1-400C allele was not associated with platelet reactivity in stable cardiovascular patients, irrespective of the platelet aggregation agonist used (collagen, ADP or arachidonic acid) or the anti-platelet drug regimen. Moreover, the Panx1-400A > C SNP did also not affect the re-occurrence of cardiac ischemic events in the same stable cardiovascular patient cohort.

Filippo Molica

Department of Pathology and Immunology

University of Geneva

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation