Metastasis is the leading cause of cancer-related death in humans. It is a complex multistep process during which individual tumour cells spread primarily through the circulatory system to colonize distant organs. Once in the circulation, tumour cells remain vulnerable, and their metastatic potential largely depends on a rapid and efficient way to escape from the blood stream by passing the endothelial barrier. Evidence has been provided that tumour cell extravasation resembles leukocyte transendothelial migration. However, it remains unclear how tumour cells interact with endothelial cells during extravasation and how these processes are regulated on a molecular level. Here we show that human and murine tumour cells induce programmed necrosis (necroptosis) of endothelial cells, which promotes tumour cell extravasation and metastasis. Treatment of mice with the receptor-interacting serine/threonine-protein kinase 1 (RIPK1)-inhibitor necrostatin-1 or endothelial-cell-specific deletion of RIPK3 reduced tumour-cell-induced endothelial necroptosis, tumour cell extravasation and metastasis. In contrast, pharmacological caspase inhibition or endothelial-cell-specific loss of caspase-8 promoted these processes. We furthermore show in vitro and in vivo that tumour-cell-induced endothelial necroptosis leading to extravasation and metastasis requires amyloid precursor protein expressed by tumour cells and its receptor, death receptor 6 (DR6), on endothelial cells as the primary mediators of these effects. Our data identify a new mechanism underlying tumour cell extravasation and metastasis, and suggest endothelial DR6-mediated necroptotic signalling pathways as targets for anti-metastatic therapies.

The vascular endothelium is constantly exposed to mechanical forces, including fluid shear stress exerted by the flowing blood. Endothelial cells can sense different flow patterns and convert the mechanical signal of laminar flow into atheroprotective signals, including eNOS activation, whereas disturbed flow in atheroprone areas induces inflammatory signaling, including NF-κB activation. How endothelial cells distinguish different flow patterns is poorly understood. Here we show that both laminar and disturbed flow activate the same initial pathway involving the mechanosensitive cation channel Piezo1, the purinergic P2Y receptor, and G/G-mediated signaling. However, only disturbed flow leads to Piezo1- and G/G-mediated integrin activation resulting in focal adhesion kinase-dependent NF-κB activation. Mice with induced endothelium-specific deficiency of Piezo1 or Gα/Gα show reduced integrin activation, inflammatory signaling, and progression of atherosclerosis in atheroprone areas. Our data identify critical steps in endothelial mechanotransduction, which distinguish flow pattern-dependent activation of atheroprotective and atherogenic endothelial signaling and suggest novel therapeutic strategies to treat inflammatory vascular disorders such as atherosclerosis.

No abstract

G-protein-coupled receptor (GPCR) expression is extensively studied in bulk cDNA, but heterogeneity and functional patterning of GPCR expression in individual vascular cells is poorly understood. Here, we perform a microfluidic-based single-cell GPCR expression analysis in primary smooth muscle cells (SMC) and endothelial cells (EC). GPCR expression is highly heterogeneous in all cell types, which is confirmed in reporter mice, on the protein level and in human cells. Inflammatory activation in murine models of sepsis or atherosclerosis results in characteristic changes in the GPCR repertoire, and we identify functionally relevant subgroups of cells that are characterized by specific GPCR patterns. We further show that dedifferentiating SMC upregulate GPCRs such as Gpr39, Gprc5b, Gprc5c or Gpr124, and that selective targeting of Gprc5b modulates their differentiation state. Taken together, single-cell profiling identifies receptors expressed on pathologically relevant subpopulations and provides a basis for the development of new therapeutic strategies in vascular diseases.

These results demonstrate that smooth muscle cells within atherosclerotic lesions can switch to a macrophage-like phenotype characterized by higher expression of inflammatory and synthetic markers genes that may further contribute to plaque progression.

Background and purpose: Resistance blood vessels are innervated by sympathetic and primary sensory nerves, which modulate vascular tone through the release of noradrenaline and calcitonin gene-related peptide (CGRP), respectively. Moreover, electrical stimulation of the perivascular sensory outflow in pithed rats results in vasodepressor responses which are mainly mediated by CGRP release. The present study has investigated the role of a 2 -adrenoceptors in the inhibition of these vasodepressor responses. Experimental approach: 144 pithed male Wistar rats were pretreated with hexamethonium (2 mg kg À1 min À1 ) followed by i.v. continuous infusions of either methoxamine (15 and 30 mg kg À1 min À1 ) or clonidine (3, 10 and 30 mg kg À1 min À1 ). Under these conditions, electrical stimulation (0.56-5.6 Hz; 50 V and 2 ms) of the spinal cord (T 9 -T 12 ) resulted in frequencydependent decreases in diastolic blood pressure. Key results: The infusion of clonidine (10 mg kg À1 min À1 ), as compared to those of methoxamine (15 or 30 mg kg À1 min À1 ), inhibited the vasodepressor responses to electrical stimulation without affecting those to i.v. bolus injections of a-CGRP (0.1-1 mg kg À1 ). This inhibition by clonidine was: (i) antagonized by 300 mg kg À1 rauwolscine (a 2A/2B/2C ), 300 and 1000 mg kg À1 BRL44408 (a 2A ), or 10 and 30 mg kg À1 MK912 (a 2C ); and (ii) unaffected by 1 ml kg À1 saline, 100 mg kg À1 BRL44408, 3000 and 10000 mg kg À1 imiloxan (a 2B ) or 3 mg kg À1 MK912. Conclusions and implications:The inhibition produced by 10 mg kg À1 min À1 clonidine on the vasodepressor (perivascular) sensory outflow in rats may be mainly mediated by prejunctional a 2A /a 2C -adrenoceptors.

British Journal of Pharmacology (2008) 154, 1160; doi:

e Vascular smooth muscle cells (vSMCs) are key in the regulation of blood pressure and the engagement of vascular pathologies, such as hypertension, arterial remodeling, and neointima formation. The role of the Rac1 GTPase in these cells remains poorly characterized. To clarify this issue, we have utilized genetically engineered mice to manipulate the signaling output of Rac1 in these cells at will using inducible, Cre-loxP-mediated DNA recombination techniques. Here, we show that the expression of an active version of the Rac1 activator Vav2 exclusively in vSMCs leads to hypotension as well as the elimination of the hypertension induced by the systemic loss of wild-type Vav2. Conversely, the specific depletion of Rac1 in vSMCs causes defective nitric oxide vasodilation responses and hypertension. Rac1, but not Vav2, also is important for neointima formation but not for hypertension-driven vascular remodeling. These animals also have allowed us to dismiss etiological connections between hypertension and metabolic disease and, most importantly, identify pathophysiological programs that cooperate in the development and consolidation of hypertensive states caused by local vascular tone dysfunctions. Finally, our results suggest that the therapeutic inhibition of Rac1 will be associated with extensive cardiovascular system-related side effects and identify pharmacological avenues to circumvent them.