The Protective Role of Natriuretic Peptide Receptor 2 against High Salt Injury in the Renal Papilla.

Mutations in natriuretic peptide receptor 2 (Npr2) gene cause a rare form of short-limbed dwarfism, but its physiological effects have not been well studied. Human and mouse genetic data suggest that Npr2 in the kidney plays a role in salt homeostasis. Herein, we described anatomic changes within renal papilla of Npr2 knockout (Npr2-/-) mice. Dramatic reduction was found in diuresis, and albuminuria was evident after administration of 1% NaCl in drinking water in Npr2-/- and heterozygous (Npr2+/-) mice compared with their wild-type (Npr2+/+) littermates. There was indication of renal epithelial damage accompanied by high numbers of red blood cells and inflammatory cells (macrophage surface glycoproteins binding to galectin-3) and an increase of renal epithelial damage marker (T-cell Ig and mucin domain 1) in Npr2-/- mice. Addition of 1% NaCl tended to increase apoptotic cells (cleaved caspase 3) in the renal papilla of Npr2-/- mice. In vitro, genetic silencing of the Npr2 abolished protective effects of C-type natriuretic peptide, a ligand for Npr2, against death of M-1 kidney epithelial cells exposed to 360 mmol/L NaCl. Finally, significantly lower levels of expression of the NPR2 protein were detected in renal samples of hypertensive compared with normotensive human subjects. Taken together, these findings suggest that Npr2 is essential to protect renal epithelial cells from high concentrations of salt and prevent kidney injury.

Innate Immune Cells Are Regulated by Axl in Hypertensive Kidney.

The balance between adaptive and innate immunity in kidney damage in salt-dependent hypertension is unclear. We investigated early renal dysfunction and the influence of Axl, a receptor tyrosine kinase, on innate immune response in hypertensive kidney in mice with lymphocyte deficiency (Rag1-/-). The data suggest that increased presence of CD11b+ myeloid cells in the medulla might explain intensified salt and water retention as well as initial hypertensive response in Rag1-/- mice. Global deletion of Axl on Rag1-/- background reversed kidney dysfunction and accumulation of myeloid cells in the kidney medulla. Chimeric mice that lack Axl in innate immune cells (in the absence of lymphocytes) significantly improved kidney function and abolished early hypertensive response. The bioinformatics analyses of Axl-related gene-gene interaction networks established tissue-specific variation in regulatory pathways. It was confirmed that complement C3 is important for Axl-mediated interactions between myeloid and vascular cells in hypertensive kidney. In summary, innate immunity is crucial for renal dysfunction in early hypertension, and is highly influenced by the presence of Axl.

Role of Axl in T-Lymphocyte Survival in Salt-Dependent Hypertension.

OBJECTIVE: Survival of immune and nonimmune cells relies on Axl, a receptor tyrosine kinase, which is implicated in hypertension. Activated T lymphocytes are involved in regulation of high blood pressure. The goal of the study was to investigate the role of Axl in T-lymphocyte functions and its contribution to salt-dependent hypertension. APPROACH AND RESULTS: We report increased apoptosis in peripheral blood from Axl(-/-) mice because of lower numbers of white blood cells mostly lymphocytes. In vitro studies showed modest reduction in interferon gamma production in Axl(-/-) type 1 T helper cells. Axl did not affect basic proliferation capacity or production of interleukin 4 in Axl(-/-) type 2 T helper cells. However, competitive repopulation of Axl(-/-) bone marrow or adoptive transfer of Axl(-/-) CD4(+) T cells to Rag1(-/-) mice showed robust effect of Axl on T lymphocyte expansion in vivo. Adoptive transfer of Axl(-/-) CD4(+) T cells was protective in a later phase of deoxycorticosterone-acetate and salt hypertension. Reduced numbers of CD4(+) T cells in circulation and in perivascular adventitia decreased vascular remodeling and increased vascular apoptosis in the late phase of hypertension. CONCLUSIONS: These findings suggest that Axl is critical for survival of T lymphocytes, especially during vascular remodeling in hypertension.

Axl modulates immune activation of smooth muscle cells in vein graft remodeling.

The pathophysiological mechanisms of the immune activation of smooth muscle cells are not well understood. Increased expression of Axl, a receptor tyrosine kinase, was recently found in arteries from patients after coronary bypass grafts. In the present study, we hypothesized that Axl-dependent immune activation of smooth muscle cells regulates vein graft remodeling. We observed a twofold decrease in intimal thickening after vascular and systemic depletion of Axl in vein grafts. Local depletion of Axl had the greatest effect on immune activation, whereas systemic deletion of Axl reduced intima due to an increase in apoptosis in vein grafts. Primary smooth muscle cells isolated from Axl knockout mice had reduced proinflammatory responses by prevention of the STAT1 pathway. The absence of Axl increased suppressor of cytokine signaling (SOCS)1 expression in smooth muscle cells, a major inhibitory protein for STAT1. Ultrasound imaging suggested that vascular depletion of Axl reduced vein graft stiffness. Axl expression determined the STAT1-SOCS1 balance in vein graft intima and progression of the remodeling. The results of this investigation demonstrate that Axl promotes STAT1 signaling via inhibition of SOCS1 in activated smooth muscle cells in vein graft remodeling.

Platelet factor 4 mediates vascular smooth muscle cell injury responses.

Activated platelets release many inflammatory molecules with important roles in accelerating vascular inflammation. Much is known about platelet and platelet-derived mediator interactions with endothelial cells and leukocytes, but few studies have examined the effects of platelets on components of the vascular wall. Vascular smooth muscle cells (VSMCs) undergo phenotypic changes in response to injury including the production of inflammatory molecules, cell proliferation, cell migration, and a decline in the expression of differentiation markers. In this study, we demonstrate that the platelet-derived chemokine platelet factor 4 (PF4/CXCL4) stimulates VSMC injury responses both in vitro and in vivo in a mouse carotid ligation model. PF4 drives a VSMC inflammatory phenotype including a decline in differentiation markers, increased cytokine production, and cell proliferation. We also demonstrate that PF4 effects are mediated, in part, through increased expression of the transcription factor Krüppel-like factor 4. Our data indicate an important mechanistic role for platelets and PF4 in VSMC injury responses both in vitro and in vivo.

Intima modifier locus 2 controls endothelial cell activation and vascular permeability.

Carotid intima formation is a significant risk factor for cardiovascular disease. C3H/FeJ (C3H/F) and SJL/J (SJL) inbred mouse strains differ in susceptibility to immune and vascular traits. Using a congenic approach we demonstrated that the Intima modifier 2 (Im2) locus on chromosome 11 regulates leukocyte infiltration. We sought to determine whether inflammation was due to changes in circulating immune cells or activation of vascular wall cells in genetically pure Im2 (C3H/F.SJL.11.1) mice. Complete blood counts showed no differences in circulating monocytes between C3H/F and C3H/F.SJL.11.1 compared with SJL mice. Aortic vascular cell adhesion molecule-1 (VCAM-1) total protein levels were dramatically increased in SJL and C3H/F.SJL.11.1 compared with C3H/F mice. Immunostaining of aortic endothelial cells (EC) showed a significant increase in VCAM-1 expression in SJL and C3H/F.SJL.11.1 compared with C3H/F under steady flow conditions. Immunostaining of EC membranes revealed a significant decrease in EC size in SJL and C3H/F.SJL.11.1 vs. C3H/F in regions of disturbed flow. Vascular permeability was significantly higher in C3H/F.SJL.11.1 compared with C3H/F. Our results indicate that Im2 regulation of leukocyte infiltration is mediated by EC inflammation and permeability. RNA sequencing and pathway analyses comparing genes in the Im2 locus to C3H/F provide insight into candidate genes that regulate vascular wall inflammation and permeability highlighting important genetic mechanisms that control vascular intima in response to injury.

Identification of a genetic locus on chromosome 11 that regulates leukocyte infiltration in mouse carotid artery.

OBJECTIVE: We demonstrated that inflammatory cells and intima-media thickening are increased in carotids exposed to low-blood flow in the SJL/J (SJL) strain compared with other mouse strains. We hypothesized that the extent of inflammation associated with intima-media thickening is a genetically regulated trait. APPROACH AND RESULTS: We performed a whole genome approach to measure leukocyte infiltration in the carotid intima as a quantitative trait in a genetic cross between C3HeB/FeJ (C3H/F) and SJL mice. Immunostaining for CD45(+) (a pan-specific leukocyte marker) was performed on carotids from C3H/F, SJL, F1, and N2 progeny to measure leukocyte infiltration. We identified a nearly significant quantitative trait locus for CD45(+) on chromosome (chr) 11 (17 cM, LOD=2.3; significance was considered at threshold P=0.05). Interval mapping showed that the CD45(+) locus on chr 11 accounted for 8% of the variation in the logarithm of odds backcross. Importantly, the CD45(+) locus colocalized with the intima-modifier 2 (Im2) locus, which controls 17% of intima variation. We created 2 Im2 congenic lines of mice (C3H/F.SJL.11.1 and C3H/F.SJL.11.2) to better understand the regulation of intima-media thickening by the chr 11 locus. The C3H/F.SJL.11.1 congenic mouse showed ≈30% of the SJL trait, confirming that CD45(+) cell infiltration contributed to the intima trait. CONCLUSIONS: We discovered a novel locus on chr 11 that controls leukocyte infiltration in the carotid. Importantly, this locus overlaps with our previously published Im2 locus on chr 11. Our study reveals a potential mechanistic relationship between leukocyte infiltration and intima-media thickening in response to decreased blood flow.

G-protein-coupled receptor kinase interacting protein-1 mediates intima formation by regulating vascular smooth muscle proliferation, apoptosis, and migration.

OBJECTIVE: The G-protein-coupled receptor kinase interacting protein-1 (GIT1) is a scaffold protein that is important for phospholipase Cγ and extracellular signal-regulated kinase 1/2 signaling induced by angiotensin II and epidermal growth factor. Because GIT1 regulates signaling by several vascular smooth muscle cell (VSMC) growth factors, we hypothesized that intima formation would be inhibited by GIT1 depletion. APPROACH AND RESULTS: Complete carotid ligation was performed on GIT1 wild-type and knockout (KO) mice. We compared changes between GIT1 wild-type and KO mice in carotid vascular remodeling, VSMC proliferation, and apoptosis in vivo and in vitro. Our data demonstrated that GIT1 deficiency significantly decreased intima formation after carotid ligation as a result of both reduced VSMC proliferation and enhanced apoptosis. To confirm the effects of GIT1 in vitro, we performed proliferation and apoptosis assays in VSMC. In mouse aortic smooth muscle cells (MASM), we found that the growth rate and [3H]-thymidine incorporation of the GIT1 KO MASM were significantly decreased compared with the wild-type MASM. Cyclin D1, which is a key cell cycle regulator, was significantly decreased in GIT1 KO cells. Serum deprivation of GIT1 KO MASM increased apoptosis 3-fold compared with wild-type MASM. Treatment of rat aortic smooth muscle cells with GIT1 small interfering RNA impaired cell migration. Both phospholipase Cγ and extracellular signal-regulated kinase 1/2 signaling were required for GIT1-dependent VSMC proliferation and migration, whereas only phospholipase Cγ was involved in GIT1-mediated VSMC apoptosis. CONCLUSIONS: GIT1 is a novel mediator of vascular remodeling by regulating VSMC proliferation, migration, and apoptosis through phospholipase Cγ and extracellular signal-regulated kinase 1/2 signaling pathways.

Ribosomal protein L17, RpL17, is an inhibitor of vascular smooth muscle growth and carotid intima formation.

BACKGROUND: Carotid intima-media thickening is associated with increased cardiovascular risk in humans. We discovered that intima formation and cell proliferation in response to carotid injury is greater in SJL/J (SJL) in comparison with C3HeB/FeJ (C3H/F) mice. The purpose of this study was to identify candidate genes contributing to intima formation. METHODS AND RESULTS: We performed microarray and bioinformatic analyses of carotid arteries from C3H/F and SJL mice. Kyoto Encyclopedia of Genes and Genomes analysis showed that the ribosome pathway was significantly up-regulated in C3H/F in comparison with SJL mice. Expression of a ribosomal protein, RpL17, was >40-fold higher in C3H/F carotids in comparison with SJL. Aortic vascular smooth muscle cells from C3H/F grew slower in comparison to SJL. To determine the role of RpL17 in vascular smooth muscle cell growth regulation, we analyzed the relationship between RpL17 expression and cell cycle progression. Cultured vascular smooth muscle cells from mice, rats, and humans showed that RpL17 expression inversely correlated with growth as shown by decreased cells in S phase and increased cells in G(0)/G(1). To prove that RpL17 acted as a growth inhibitor in vivo, we used pluronic gel delivery of RpL17 small interfering RNA to C3H/F carotid arteries. This resulted in an 8-fold increase in the number of proliferating cells. Furthermore, following partial carotid ligation in SJL mice, RpL17 expression in the intima and media decreased, but the number of proliferating cells increased. CONCLUSIONS: RpL17 acts as a vascular smooth muscle cell growth inhibitor (akin to a tumor suppressor) and represents a potential therapeutic target to limit carotid intima-media thickening.

Immune modulation of vascular resident cells by Axl orchestrates carotid intima-media thickening.

Cellular mechanisms of carotid intima-media thickening (IMT) are largely unknown. The receptor tyrosine kinase Axl is essential for function of both bone marrow (BM) and non-BM cells. We studied the mechanisms by which Axl expression in BM-derived cells (compared with non-BM-derived cells) mediates carotid IMT. Partial ligation of the left carotid artery resulted in a similar carotid blood flow reduction in Axl chimeras. Neither irradiation nor bone marrow transplantation had any effect on the 40% difference in carotid IMT between Axl genotypes. Axl-dependent survival is very important for intimal leukocytes; however, Axl expression in BM cells contributes to <30% of carotid IMT. Axl in non-BM cells has a greater effect on carotid remodeling. Expression of Axl in non-BM cells is crucial for the up-regulation of several key proinflammatory signals (eg, IL-1) in the carotid. We found that Axl is involved in immune activation of cultured smooth muscle cells and in immune heterogeneity of medial cells (measured by major histocompatibility complex class II) after carotid injury. Finally, a lack of Axl in non-BM cells increased collagen Iα expression, which may play a critical role in carotid remodeling. Our data suggest that Axl contributes to carotid remodeling not only by inhibition of apoptosis but also via regulation of immune heterogeneity of vascular cells, cytokine/chemokine expression, and extracellular matrix remodeling.

Genetic locus on mouse chromosome 7 controls elevated heart rate.

Elevated heart rate (HR) is a risk factor for cardiovascular diseases. The goal of the study was to map HR trait in mice using quantitative trait locus (QTL) analysis followed by genome-wide association (GWA) analysis. The first approach provides mapping power and the second increases genome resolution. QTL analyses were performed in a C3HeB×SJL backcross. HR and systolic blood pressure (SBP) were measured by the tail-cuff plethysmography. HR was ∼80 beats/min higher in SJL compared with C3HeB. There was a wide distribution of the HR (536-763 beats/min) in N2 mice. We discovered a highly significant QTL (logarithm of odds = 6.7, P < 0.001) on chromosome 7 (41 cM) for HR in the C3HeB×SJL backcross. In the Hybrid Mouse Diversity Panel (58 strains, n = 5-6/strain) we found that HR (beats/min) ranged from 546 ± 12 in C58/J to 717 ± 7 in MA/MyJ mice. SBP (mmHg) ranged from 99 ± 6 in strain I/LnJ to 151 ± 4 in strain BXA4/PgnJ. GWA analyses were done using the HMDP, which revealed a locus (64.2-65.1 Mb) on chromosome 7 that colocalized with the QTL for elevated HR found in the C3HeB×SJL backcross. The peak association was observed for 17 SNPs that are localized within three GABA(A) receptor genes. In summary, we used a combined genetic approach to fine map a novel elevated HR locus on mouse chromosome 7.

Hybrid mouse diversity panel: a panel of inbred mouse strains suitable for analysis of complex genetic traits.

We have developed an association-based approach using classical inbred strains of mice in which we correct for population structure, which is very extensive in mice, using an efficient mixed-model algorithm. Our approach includes inbred parental strains as well as recombinant inbred strains in order to capture loci with effect sizes typical of complex traits in mice (in the range of 5% of total trait variance). Over the last few years, we have typed the hybrid mouse diversity panel (HMDP) strains for a variety of clinical traits as well as intermediate phenotypes and have shown that the HMDP has sufficient power to map genes for highly complex traits with resolution that is in most cases less than a megabase. In this essay, we review our experience with the HMDP, describe various ongoing projects, and discuss how the HMDP may fit into the larger picture of common diseases and different approaches.

Novel tyrosine kinase signaling pathways: implications in vascular remodeling.

PURPOSE OF REVIEW: This review summarizes the recent advances in molecular mechanisms by which five classes of receptor tyrosine kinases (RTKs) contribute to vascular remodeling. RECENT FINDINGS: Recent findings have expanded our knowledge regarding RTK regulation. In particular, G-protein-coupled receptors, mineralocorticoid receptors, mechanical and oxidative stresses transactivate RTKs. These receptors are highly interactive with many downstream targets (including tyrosine kinases and other RTKs) and function as key regulatory nodes in a dynamic signaling network. Interactions between vascular and nonvascular (immune and neuronal) cells are controlled by RTKs in vascular remodeling. Inhibition of RTKs could be an advantageous therapeutic strategy for vascular disorders. SUMMARY: RTK-dependent signaling is important for regulation of key functions during vascular remodeling. However, current challenges are related to integration of the data on multiple RTKs in vascular pathology.

Axl-dependent signalling: a clinical update.

Axl is a receptor tyrosine kinase that was originally cloned from cancer cells. Axl belongs to the TAM (Tyro3, Axl and Mertk) family of receptor tyrosine kinases. Gas6 (growth-arrest-specific protein 6) is a ligand for Axl. Activation of Axl protects cells from apoptosis, and increases migration, aggregation and growth through multiple downstream pathways. Up-regulation of the Gas6/Axl pathway is more evident in pathological conditions compared with normal physiology. Recent advances in Axl receptor biology are summarized in the present review. The emphasis is given to translational aspects of Axl-dependent signalling under pathological conditions. In particular, inhibition of Axl reduces tumorigenesis and prevents metastasis as well. Axl-dependent signals are important for the progression of cardiovascular diseases. In contrast, deficiency of Axl in innate immune cells contributes to the pathogenesis of autoimmune disorders. Current challenges in Axl biology are related to the functional interactions of Axl with other members of the TAM family or other tyrosine kinases, mechanisms of ligand-independent activation, inactivation of the receptor and cell-cell interactions (with respect to immune cells) in chronic diseases.

Role of PDE10A in vascular smooth muscle cell hyperplasia and pathological vascular remodelling.

AIMS: Intimal hyperplasia is a common feature of vascular remodelling disorders. Accumulation of synthetic smooth muscle cell (SMC)-like cells is the main underlying cause. Current therapeutic approaches including drug-eluting stents are not perfect due to the toxicity on endothelial cells and novel therapeutic strategies are needed. Our preliminary screening for dysregulated cyclic nucleotide phosphodiesterases (PDEs) in growing SMCs revealed the alteration of PDE10A expression. Herein, we investigated the function of PDE10A in SMC proliferation and intimal hyperplasia both in vitro and in vivo. METHODS AND RESULTS: RT-qPCR, immunoblot, and in situ proximity ligation assay were performed to determine PDE10A expression in synthetic SMCs and injured vessels. We found that PDE10A mRNA and/or protein levels are up-regulated in cultured SMCs upon growth stimulation, as well as in intimal cells in injured mouse femoral arteries. To determine the cellular functions of PDE10A, we focused on its role in SMC proliferation. The anti-mitogenic effects of PDE10A on SMCs were evaluated via cell counting, BrdU incorporation, and flow cytometry. We found that PDE10A deficiency or inhibition arrested the SMC cell cycle at G1-phase with a reduction of cyclin D1. The anti-mitotic effect of PDE10A inhibition was dependent on cGMP-dependent protein kinase Iα (PKGIα), involving C-natriuretic peptide (CNP) and particulate guanylate cyclase natriuretic peptide receptor 2 (NPR2). In addition, the effects of genetic depletion and pharmacological inhibition of PDE10A on neointimal formation were examined in a mouse model of femoral artery wire injury. Both PDE10A knockout and inhibition decreased injury-induced intimal thickening in femoral arteries by at least 50%. Moreover, PDE10A inhibition decreased ex vivo remodelling of cultured human saphenous vein segments. CONCLUSIONS: Our findings indicate that PDE10A contributes to SMC proliferation and intimal hyperplasia at least partially via antagonizing CNP/NPR2/cGMP/PKG1α signalling and suggest that PDE10A may be a novel drug target for treating vascular occlusive disease.

Bcr kinase activation by angiotensin II inhibits peroxisome-proliferator-activated receptor gamma transcriptional activity in vascular smooth muscle cells.

Bcr is a serine/threonine kinase activated by platelet-derived growth factor that is highly expressed in the neointima after vascular injury. Here, we demonstrate that Bcr is an important mediator of angiotensin (Ang) II and platelet-derived growth factor-mediated inflammatory responses in vascular smooth muscle cells (VSMCs). Among transcription factors that might regulate Ang II-mediated inflammatory responses we found that ligand-mediated peroxisome proliferator-activated receptor (PPAR)gamma transcriptional activity was significantly decreased by Ang II. Ang II increased Bcr expression and kinase activity. Overexpression of Bcr significantly inhibited PPARgamma activity. In contrast, knockdown of Bcr using Bcr small interfering RNA and a dominant-negative form of Bcr (DN-Bcr) reversed Ang II-mediated inhibition of PPARgamma activity significantly, suggesting the critical role of Bcr in Ang II-mediated inhibition of PPARgamma activity. Point-mutation and in vitro kinase analyses showed that PPARgamma was phosphorylated by Bcr at serine 82. Overexpression of wild-type Bcr kinase did not inhibit ligand-mediated PPARgamma1 S82A mutant transcriptional activity, indicating that Bcr regulates PPARgamma activity via S82 phosphorylation. DN-Bcr and Bcr small interfering RNA inhibited Ang II-mediated nuclear factor kappaB activation in VSMCs. DN-PPARgamma reversed DN-Bcr-mediated inhibition of nuclear factor kappaB activation, suggesting that PPARgamma is downstream from Bcr. Intimal proliferation in low-flow carotid arteries was decreased in Bcr knockout mice compared with wild-type mice, suggesting the critical role of Bcr kinase in VSMC proliferation in vivo, at least in part, via regulating PPARgamma/nuclear factor kappaB transcriptional activity.

Genetic modifier loci linked to intima formation induced by low flow in the mouse carotid.

OBJECTIVE: Previously we found dramatic strain-dependent differences in a low flow model of vascular remodeling. Specifically, intima formation in the left common carotid artery was approximately 30-fold greater in SJL compared to C3HeB/Fe (C3H/F) mice. We hypothesized that a few genes control intima formation in response to low flow. A C3H/F and SJL backcross resulted in broad range of N2 intima phenotypes. METHODS AND RESULTS: Using genome-wide scan we identified two highly significant quantitative trait loci (QTLs) for intima, Im1 (intima modifier 1 locus) on chromosome 2 (Chr2; 77.6 cM, LOD=6.4), and Im2 on Chr11 (17 cM, LOD=5.3). One significant QTL Im3 was found on Chr18 (6 cM, LOD=3.0), and two suggestive QTLs (LOD=1.5 and 1.8) were identified on Chr7 and Chr17, respectively. Interestingly, the intima/media ratio trait mapped to the same QTLs as the intima trait. Haplotype mapping predicted 40 candidate genes. Six of these genes contained SNPs that differed between C3H/F and SJL. CONCLUSIONS: We have successfully mapped 3 QTLs (Im1, Im2, and Im3) that are associated with carotid intima formation in response to low blood flow. These results may be important in identifying genes that influence carotid intima-media thickening and predict cardiovascular disease in humans.

Fibronectin is an important regulator of flow-induced vascular remodeling.

OBJECTIVE: Fibronectin is an important regulator of cell migration, differentiation, growth, and survival. Our data show that fibronectin also plays an important role in regulating extracellular matrix (ECM) remodeling. Fibronectin circulates in the plasma and is also deposited into the ECM by a cell dependent process. To determine whether fibronectin affects vascular remodeling in vivo, we asked whether the fibronectin polymerization inhibitor, pUR4, inhibits intima-media thickening, and prevents excess ECM deposition in arteries using a mouse model of vascular remodeling. METHODS AND RESULTS: To induce vascular remodeling, partial ligation of the left external and internal carotid arteries was performed in mice. pUR4 and the control peptide were applied periadventitially in pluronic gel immediately after surgery. Animals were euthanized 7 or 14 days after surgery. Morphometric analysis demonstrated that the pUR4 fibronectin inhibitor reduced carotid intima (63%), media (27%), and adventitial thickening (40%) compared to the control peptide (III-11C). Treatment with pUR4 also resulted in a dramatic decrease in leukocyte infiltration into the vessel wall (80%), decreased ICAM-1 and VCAM-1 levels, inhibited cell proliferation (60% to 70%), and reduced fibronectin and collagen I accumulation in the vessel wall. In addition, the fibronectin inhibitor prevented SMC phenotypic modulation, as evidenced by the maintenance of smooth muscle (SM) alpha-actin and SM myosin heavy chain levels in medial cells. CONCLUSIONS: These data are the first to demonstrate that fibronectin plays an important role in regulating the vascular remodeling response. Collectively, these data suggest a therapeutic benefit of periadventitial pUR4 in reducing pathological vascular remodeling.

Natriuretic Peptide Receptor 2 Locus Contributes to Carotid Remodeling.

Background Carotid artery intima/media thickness (IMT) is a hallmark trait associated with future cardiovascular events. The goal of this study was to map new genes that regulate carotid IMT by genome-wide association. Methods and Results We induced IMT by ligation procedure of the left carotid artery in 30 inbred mouse strains. Histologic reconstruction revealed significant variation in left carotid artery intima, media, adventitia, external elastic lamina volumes, intima-to-media ratio, and (intima+media)/external elastic lamina percent ratio in inbred mice. The carotid remodeling trait was regulated by distinct genomic signatures with a dozen common single-nucleotide polymorphisms associated with left carotid artery intima volume, intima-to-media ratio, and (intima+media)/external elastic lamina percent ratio. Among genetic loci on mouse chromosomes 1, 4, and 12, there was natriuretic peptide receptor 2 (Npr2), a strong candidate gene. We observed that only male, not female, mice heterozygous for a targeted Npr2 deletion (Npr2+/-) exhibited defective carotid artery remodeling compared with Npr2 wild-type (Npr2+/+) littermates. Fibrosis in carotid IMT was significantly increased in Npr2+/- males compared with Npr2+/- females or Npr2+/+ mice. We also detected decreased Npr2 expression in human atherosclerotic plaques, similar to that seen in studies in Npr2+/- mice. Conclusions We found that components of carotid IMT were regulated by distinct genetic factors. We also showed a critical role for Npr2 in genetic regulation of vascular fibrosis associated with defective carotid remodeling.

Strain-selective efficacy of sacubitril/valsartan on carotid fibrosis in response to injury in two inbred mouse strains.

BACKGROUND AND PURPOSE: Sacubitril/valsartan (Sac/val) is more effective than valsartan in lowering BP and mortality in patients with heart failure. Here, we proposed that Sac/val treatment would be more effective in preventing pathological vascular remodelling in 129X1/SvJ (129X1), than in C57BL/6J (B6) inbred mice. EXPERIMENTAL APPROACH: Sac/val (60 mg·kg-1 ·day-1 ) and valsartan (27 mg·kg-1 ·day-1 ) were given as prophylactic or therapeutic treatments, to 129X1 or B6 mice with carotid artery ligation for 14 days. Blood flow was measured by ultrasound. Ex vivo, carotid tissue was analysed with histological and morphometric techniques, together with RNA sequencing and gene ontology. KEY RESULTS: Sac/val was more effective than valsartan in lowering BP in 129X1 compared with B6 mice. Liver expression of CYP2C9 and plasma cGMP levels were similar across treatments. A reduction in carotid thickening after prophylactic treatment with valsartan or Sac/val also resulted in significant arterial shrinkage in B6 mice. In 129X1 mice, Sac/val and prophylactic treatment with valsartan had no effect on carotid thickening but preserved carotid size. BP lowering significantly correlated with a decline in carotid stiffness (R2  = .37, P = .0096) in 129X1 but not in B6 mice. The gene expression signature associated with hyalurononglucosaminidase activity was down-regulated in injured arteries after both regimens of Sac/val only in 129X1 mice. Administration of Sac/val but not valsartan significantly reduced deposition of hyaluronic acid and carotid fibrosis in 129X1 mice. CONCLUSION AND IMPLICATIONS: These results underscore the importance of the genetic background in the efficacy of the Sac/val on vascular fibrosis.