Morphological assessment of renal arteries after radiofrequency catheter-based sympathetic denervation in a porcine model.

OBJECTIVES: Catheter-based renal artery denervation has been successfully introduced as alternative treatment for patients suffering from drug-resistant essential hypertension. However, the local morphological changes within the vessel wall accompanying this technique remain elusive and we sought to characterize these by utilizing the simplicity radiofrequency catheter approach. METHODS: Following treatment of seven pigs, renal arteries were assigned to either the acute (n  =  6), subacute (10-day follow-up, n  =  6) or control (untreated, n  =  2) group. At follow-up blood analysis, final angiography and optical coherence tomography (OCT)-imaging of the treated arteries were performed and renal arteries and kidneys were processed for histopathology and immunohistochemistry. RESULTS: Radiofrequency-derived energy application to the vessel wall induced transmural tissue coagulation and loss of endothelium resulting in local thrombus formation also detectable by OCT. At 10 days, the luminal surface was almost completely re-endothelialized. Mural wall damage was replaced by fibrotic tissue and the adventitial layer showed strong inflammatory infiltration including vasculogenesis. Remnant autonomic nerve fascicles within the lesion segments of the subacute group displayed enhanced vacuolic degeneration and an impaired neurofilament protein immunostaining pattern. Examination of the kidneys revealed no abnormalities and blood parameters remained within the physiological range. CONCLUSION: Catheter-based application of radiofrequency energy resulted in circumscribed transmural injury within the arterial wall affecting autonomic nerve fascicles delayed to treatment. Acute loss of endothelialization resulted in thrombus formation leaving kidney perfusion apparently unimpaired.

Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases.

Blood neutrophils provide the first line of defense against pathogens but have also been implicated in thrombotic processes. This dual function of neutrophils could reflect an evolutionarily conserved association between blood coagulation and antimicrobial defense, although the molecular determinants and in vivo significance of this association remain unclear. Here we show that major microbicidal effectors of neutrophils, the serine proteases neutrophil elastase and cathepsin G, together with externalized nucleosomes, promote coagulation and intravascular thrombus growth in vivo. The serine proteases and extracellular nucleosomes enhance tissue factor- and factor XII-dependent coagulation in a process involving local proteolysis of the coagulation suppressor tissue factor pathway inhibitor. During systemic infection, activation of coagulation fosters compartmentalization of bacteria in liver microvessels and reduces bacterial invasion into tissue. In the absence of a pathogen challenge, neutrophil-derived serine proteases and nucleosomes can contribute to large-vessel thrombosis, the main trigger of myocardial infarction and stroke. The ability of coagulation to suppress pathogen dissemination indicates that microvessel thrombosis represents a physiological tool of host defense.

Contribution of cyclooxygenase-1 to thromboxane formation, platelet-vessel wall interactions and atherosclerosis in the ApoE null mouse.

OBJECTIVE: Prostaglandin and thromboxane (TXA(2)) generation is increased in atherosclerosis. Studies with selective inhibitors attribute the enhanced prostacyclin (PGI(2)) generation to both cyclooxygenase-1 (COX-1) and COX-2 whereas the increased TXA(2) generation reflects platelet COX-1 expression. However, TXA(2) formation remains elevated in patients with cardiovascular disease on doses of aspirin that fully suppress platelet COX-1, suggesting other tissue sources for TXA(2) formation. Disruption of the thromboxane receptor gene suppresses the development of atherosclerosis. Notwithstanding this, the role of COX-1 in atherosclerosis is unclear, as it is widely distributed and contributes to a number of products, including those that potentially contribute to the resolution of inflammation. METHODS AND RESULTS: We examined the role of COX-1 on prostaglandin generation, development of atherosclerosis and platelet-vessel wall interactions in the apoE(-/-) murine model by disrupting the COX-1 gene. ApoE(-/-)/COX-1(+/+), ApoE(-/-)/COX-1(+/-) and ApoE(-/-)/COX-1(-/-), were administered a 1% cholesterol diet for 8 weeks. Stable urinary metabolites of PGI(2) and TXA(2), which were markedly increased in the ApoE(-/-)/COX-1(+/+) were reduced by disruption of COX-1. Deletion of one or both copies of the COX-1 gene suppressed lesion formation. Assessment of platelet-vessel wall interactions by intravital microscopy showed a significant decrease in firm adhesion of platelets in the apoE/COX-1 double knockout (DKO). CONCLUSION: COX-1 contributes to the enhanced formation of both PGI(2) and TXA(2) in atherosclerosis, and to the development of the disease. Non-platelet sources of COX-1 and TXA(2) that are inaccessible to standard doses of aspirin may contribute to the development of atherosclerosis.

Extracellular RNA constitutes a natural procoagulant cofactor in blood coagulation.

Upon vascular injury, locally controlled haemostasis prevents life-threatening blood loss and ensures wound healing. Intracellular material derived from damaged cells at these sites will become exposed to blood components and could contribute to blood coagulation and pathological thrombus formation. So far, the functional and mechanistic consequences of this concept are not understood. Here, we present in vivo and in vitro evidence that different forms of eukaryotic and prokaryotic RNA serve as promoters of blood coagulation. Extracellular RNA was found to augment (auto-)activation of proteases of the contact phase pathway of blood coagulation such as factors XII and XI, both exhibiting strong RNA binding. Moreover, administration of exogenous RNA provoked a significant procoagulant response in rabbits. In mice that underwent an arterial thrombosis model, extracellular RNA was found associated with fibrin-rich thrombi, and pretreatment with RNase (but not DNase) significantly delayed occlusive thrombus formation. Thus, extracellular RNA derived from damaged or necrotic cells particularly under pathological conditions or severe tissue damage represents the long sought natural "foreign surface" and provides a procoagulant cofactor template for the factors XII/XI-induced contact activation/amplification of blood coagulation. Extracellular RNA thereby reveals a yet unrecognized target for antithrombotic intervention, using RNase or related therapeutic strategies.

Platelets secrete stromal cell-derived factor 1alpha and recruit bone marrow-derived progenitor cells to arterial thrombi in vivo.

The accumulation of smooth muscle and endothelial cells is essential for remodeling and repair of injured blood vessel walls. Bone marrow-derived progenitor cells have been implicated in vascular repair and remodeling; however, the mechanisms underlying their recruitment to the site of injury remain elusive. Here, using real-time in vivo fluorescence microscopy, we show that platelets provide the critical signal that recruits CD34+ bone marrow cells and c-Kit+ Sca-1+ Lin- bone marrow-derived progenitor cells to sites of vascular injury. Correspondingly, specific inhibition of platelet adhesion virtually abrogated the accumulation of both CD34+ and c-Kit+ Sca-1+ Lin- bone marrow-derived progenitor cells at sites of endothelial disruption. Binding of bone marrow cells to platelets involves both P-selectin and GPIIb integrin on platelets. Unexpectedly, we found that activated platelets secrete the chemokine SDF-1alpha, thereby supporting further primary adhesion and migration of progenitor cells. These findings establish the platelet as a major player in the initiation of vascular remodeling, a process of fundamental importance for vascular repair and pathological remodeling after vascular injury.

Platelet adhesion via glycoprotein IIb integrin is critical for atheroprogression and focal cerebral ischemia: an in vivo study in mice lacking glycoprotein IIb.

BACKGROUND: The platelet glycoprotein (GP) IIb/IIIa integrin binds to fibrinogen and thereby mediates platelet aggregation. Here, we addressed the role of GP IIb for platelet adhesion and determined the relevance of platelet GP IIb for the processes of atherosclerosis and cerebral ischemia-reperfusion (I/R) injury. METHODS AND RESULTS: GP IIb(-/-) mice were generated and bred with ApoE(-/-) animals to create GP IIb(-/-)ApoE(-/-) mice. Platelet adhesion to the mechanically injured or atherosclerotic vessel wall was monitored by in vivo video fluorescence microscopy. In the presence of GP IIb, vascular injury and early atherosclerosis induced platelet adhesion in the carotid artery (CA). In contrast, platelet adhesion was significantly reduced in the absence of GP IIb integrin (P<0.05). To address the contribution of platelet GP IIb to atheroprogression, we determined atherosclerotic lesion formation in the CA and aortic arch (AA) of GP IIb(+/+)ApoE(-/-) or GP IIb(-/-)ApoE(-/-) mice. Interestingly, the absence of GP IIb attenuated lesion formation in CA and AA, indicating that platelets, via GP IIb, contribute substantially to atherosclerosis. Next, we assessed the implication of GP IIb for cerebral I/R injury. We observed that after occlusion of the middle cerebral artery, the cerebral infarct size was drastically reduced in mice lacking GP IIb compared with wild-types. CONCLUSIONS: These findings show for the first time in vivo that GP IIb not only mediates platelet aggregation but also triggers platelet adhesion to exposed extracellular matrices and dysfunctional endothelial cells. In a process strictly involving GP IIb, platelets, which are among the first blood cells to arrive at the scene of endothelial dysfunction, contribute essentially to atherosclerosis and cerebral I/R injury.