Atrial natriuretic peptide (ANP) is an important regulator of arterial blood pressure. The mechanisms mediating its hypotensive effects are complex and involve the inhibition of the sympathetic and renin-angiotensin-aldosterone (RAA) systems, increased diuresis/natriuresis, vasodilation, and enhanced vascular permeability. In particular, the contribution of the direct vasodilating effect of ANP to the hypotensive actions remains controversial, because variable levels of the ANP receptor, guanylyl cyclase A (GC-A), are expressed in different vascular beds. The objective of our study was to determine whether a selective deletion of GC-A in vascular smooth muscle would affect the hypotensive actions of ANP. We first created a mutant allele of mouse GC-A by flanking a required exon with loxP sequences. Crossing floxed GC-A with SM22-Cre transgene mice expressing Cre recombinase in smooth muscle cells (SMC) resulted in mice in which vascular GC-A mRNA expression was reduced by ≈80%. Accordingly, the relaxing effects of ANP on isolated vessels from these mice were abolished; despite this fact, chronic arterial blood pressure of awake SMC GC-A KO mice was normal. Infusion of ANP caused immediate decreases in blood pressure in floxed GC-A but not in SMC GC-A knockout mice. Furthermore, acute vascular volume expansion, which causes release of cardiac ANP, did not affect resting blood pressure of floxed GC-A mice, but rapidly and significantly increased blood pressure of SMC GC-A knockout mice. We conclude that vascular GC-A is dispensable in the chronic and critical in the acute moderation of arterial blood pressure by ANP.

Rationale:: Endothelial dysfunction and atherosclerosis are chronic inflammatory diseases characterized by activation of the innate and acquired immune system. Specialized protein receptors of the innate immune system recognize products of microorganisms and endogenous ligands such as nucleic acids. Toll-like receptor 3 (TLR3), for example, detects long double-stranded RNA and is abundantly expressed in endothelial cells. Whether innate immunity contributes to atherogenic mechanisms in endothelial cells is poorly understood. Objective:: We sought to determine the effects of TLR3 activation in endothelial cells. Methods and Results:: We first investigated whether stimulation of TLR3 influences endothelial biology in mice. Intravenous injection of polyinosine polycytidylic acid, a synthetic double-stranded RNA analog and TLR3 ligand, impaired endothelium-dependent vasodilation, increased vascular production of reactive oxygen species, and reduced reendothelialization after carotid artery injury in wild-type mice compared with controls but had no effect in TLR3 ^−/− animals. TLR3 stimulation not only induced endothelial dysfunction but also enhanced the formation of atherosclerotic plaques in apolipoprotein E–deficient mice. In vitro incubation of endothelial cells with polyinosine polycytidylic acid induced production of the proinflammatory cytokines interleukin-8 and interferon-γ–induced protein 10, increased formation of reactive oxygen species, diminished proliferation, and increased apoptosis, which suggests that endothelial cells are able to directly detect and respond to TLR3 ligands. Neutralization of interleukin-8 and interferon-γ–induced protein 10 antagonizes the observed negative effects of polyinosine polycytidylic acid. We found elevated levels of circulating endothelial progenitor cells in polyinosine polycytidylic acid–treated mice, although they displayed increased endothelial dysfunction. Stimulation of TLR3 in cultured endothelial progenitor cells, however, led to increased formation of reactive oxygen species, increased apoptosis, and reduced migration. Injection of endothelial progenitor cells that had been incubated with polyinosine polycytidylic acid ex vivo hindered reendothelialization after carotid artery injury. Therefore, endothelial progenitor cell function was affected by TLR3 stimulation. Finally, apolipoprotein E–deficient/TLR3-deficient mice exhibited improved endothelial function compared with apolipoprotein E–deficient/TLR3 ^+/+ littermates. Conclusions:: Immunorecognition of long double-stranded RNA by endothelial cells may be an important mechanism involved in endothelial cell activation and development of endothelial dysfunction.

Atherosclerosis is the most common cause for cardiovascular diseases and is based on endothelial dysfunction. A growing body of evidence suggests the contribution of bone marrow–derived endothelial progenitor cells, monocytic cells, and mature endothelial cells to vessel formation and endothelial rejuvenation. To this day, various subsets of these endothelial-regenerating cells have been identified according to cellular origin, phenotype, and properties in vivo and in vitro. However, the definition and biology, especially of endothelial progenitor cells, is complex and under heavy debate. In this review, we focus on current definitions of endothelial progenitor cells, highlight the clinical relevance of endothelial-regenerating cells, and provide new insights into cell-cell interactions involved in endothelial cell rejuvenation.

Aims We assessed aortic valves from patients with non-rheumatic aortic valve stenosis (AS) and with degenerative aortic valve bioprostheses (BP) for the presence of progenitor cell and leukocyte subtype-specific markers. Methods and results Diseased valve probes from a total of 87 patients (60 AS and 27 BP) were studied. We assessed presence and localization of endothelial progenitor cells (EPCs: CD34, CD133), dendritic cells (DCs: S100), T-lymphocytes (CD3), and macrophages (CD68) by immunohistochemical and morphometric analyses. In the majority of valves, we detected cell-bound signals of CD34 (48% of AS, 74% of BP, respectively), CD133 (58%/81%), S100 (58%/93%), CD3 (62%/81%), and CD68 (78%/93%). Labelled cells were predominantly localized within the valvular fibrosa. As key results, frequency of EPCs, DCs, macrophages, and lymphocytes was found significantly higher in BP when compared with AS (CD34: 19.2±23.2 vs. 5.7±13.0%; CD133: 13.7±12.4 vs. 5.5±8.3%; S100: 15.2±12.2 vs. 5.7±8.9%; CD3: 3.3±2.7 vs. 1.1±1.4%; CD68: 35.3±26.6 vs. 3.4±4.1%; each P≤0.001). Conclusion EPCs and DCs were detected in a large collective of degenerative aortic valves, more frequently in bioprostheses than in native cusps. Aortoluminal presence of these primarily extravalvular cells co-localized with inflammatory cells is a novel key feature involved in aortic valve degeneration.

Toll-like receptors (TLR) of the innate immune system have been closely linked with the development of atherosclerotic lesions. TLR9 is activated by unmethylated CpG motifs within ssDNA, but also by CpG motifs in nucleic acids released during vascular apoptosis and necrosis. The role of TLR9 in vascular disease remains controversial and we sought to investigate the effects of a proinflammatory TLR9 stimulation in mice. TLR9-stimulation with high dose CpG ODN at concentrations between 6.25nM to 30nM induced a significant proinflammatory cytokine response in mice. This was associated with impaired reendothelialization upon acute denudation of the carotid and increased numbers of circulating endothelial microparticles, as a marker for amplified endothelial damage. Chronic TLR9 agonism in apolipoprotein E-deficient (ApoE^-/-) mice fed a cholesterol-rich diet increased aortic production of reactive oxygen species, the number of circulating endothelial microparticles, circulating sca-1/flk-1 positive cells, and most importantly augmented atherosclerotic plaque formation when compared to vehicle treated animals. Importantly, high concentrations of CpG ODN are required for these proatherogenic effects. Systemic stimulation of TLR9 with high dose CpG ODN impaired reendothelialization upon acute vascular injury and increased atherosclerotic plaque development in ApoE^-/- mice. Further studies are necessary to fully decipher the contradictory finding of TLR9 agonism in vascular biology.