Macrophage Notch Ligand Delta-Like 4 Promotes Vein Graft Lesion Development, Implications for the Treatment of Vein Graft Failure

Objective—Despite its large clinical impact, the underlying mechanisms for vein graft failure remain obscure and no effective therapeutic solutions are available. We tested the hypothesis that Notch signaling promotes vein graft disease. Approach and Results—We used 2 biotherapeutics for Delta-like ligand 4 (Dll4), a Notch ligand: (1) blocking antibody and (2) macrophage- or endothelial cell (EC)–targeted small-interfering RNA. Dll4 antibody administration for 28 days inhibited vein graft lesion development in low-density lipoprotein (LDL) receptor-deficient (Ldlr−/−) mice, and suppressed macrophage accumulation and macrophage expression of proinflammatory M1 genes. Dll4 antibody treatment for 7 days after grafting also reduced macrophage burden at day 28. Dll4 silencing via macrophage-targeted lipid nanoparticles reduced lesion development and macrophage accumulation, whereas EC-targeted Dll4 small-interfering RNA produced no effects. Gain-of-function and loss-of-function studies suggested in vitro that Dll4 induces proinflammatory molecules in macrophages. Macrophage Dll4 also stimulated smooth muscle cell proliferation and migration and suppressed their differentiation. Conclusions—These results suggest that macrophage Dll4 promotes lesion development in vein grafts via macrophage activation and crosstalk between macrophages and smooth muscle cells, supporting the Dll4–Notch axis as a novel therapeutic target.United States. National Institutes of Health (R01HL107550)American Heart Association (0655878T)American Heart Association (12GRNT9510001)American Heart Association (12GRNT1207025)Good Samaritan FoundationShapiro Family Foundatio

Association of <i>ATP1A1</i> and <i>Dear</i> Single-Nucleotide Polymorphism Haplotypes With Essential Hypertension

Essential hypertension remains a major risk factor for cardiovascular and cerebrovascular diseases. As a complex multifactorial disease, elucidation of susceptibility loci remains elusive. ATP1A1 and Dear are candidate genes for 2 closely linked rat chromosome-2 blood pressure quantitative trait loci. Because corresponding human syntenic regions are on different chromosomes, investigation of ATP1A1 (chromosome [chr]-1p21) and Dear (chr-4q31.3) facilitates genetic analyses of each blood pressure quantitative trait locus in human hypertension. Here we report the association of human ATP1A1 ( P <0.000005) and Dear ( P <0.03) with hypertension in a relatively isolated, case/control hypertension cohort from northern Sardinia by single-nucleotide polymorphism haplotype analysis. Sex-specific haplotype analyses detected stronger association of both loci with hypertension in males than in females. Haplotype trend-regression analyses support ATP1A1 and Dear as independent susceptibility loci and reveal haplotype-specific association with hypertension and normotension, thus delineating haplotype-specific subsets of hypertension. Although investigation in other cohorts needs to be performed to determine genetic effects in other populations, haplotype subtyping already allows systematic stratification of susceptibility and, hence, clinical heterogeneity, a prerequisite for unraveling the polygenic etiology and polygene–environment interactions in essential hypertension. As hypertension susceptibility genes, coexpression of ATP1A1 and Dear in both renal tubular cells and vascular endothelium suggest a cellular pathogenic scaffold for polygenic mechanisms of hypertension, as well as the hypothesis that ATP1A1 and/or Dear could contribute to the known renal and vascular endothelial dysfunction associated with essential (polygenic) hypertension

Analysis of CD45- [CD34+/KDR+] endothelial progenitor cells as juvenile protective factors in a rat model of ischemic-hemorrhagic stroke.

Identification of juvenile protective factors (JPFs) which are altered with age and contribute to adult-onset diseases could identify novel pathways for reversing the effects of age, an accepted non-modifiable risk factor to adult-onset diseases. Since endothelial progenitor cells (EPCs) have been observed to be altered in stroke, hypertension and hypercholesterolemia, said EPCs are candidate JPFs for adult-onset stroke. A priori, if EPC aging plays a 'master-switch JPF-role' in stroke pathogenesis, juvenile EPC therapy alone should delay stroke-onset. Using a hypertensive, transgenic-hyperlipidemic rat model of spontaneous ischemic-hemorrhagic stroke, spTg25, we tested the hypothesis that freshly isolated juvenile EPCs are JPFs that can attenuate stroke progression and delay stroke onset.FACS analysis revealed that CD45- [CD34+/KDR+] EPCs decrease with progression to stroke in spTg25 rats, exhibit differential expression of the dual endodthelin-1/VEGFsp receptor (DEspR) and undergo differential DEspR-subtype specific changes in number and in vitro angiogenic tube-incorporation. In vivo EPC infusion of male, juvenile non-expanded cd45-[CD34+/KDR+] EPCs into female stroke-prone rats prior to stroke attenuated progression and delayed stroke onset (P<0.003). Detection of Y-chromosome DNA in brain microvessels of EPC-treated female spTg25 rats indicates integration of male EPCs into female rat brain microvessels. Gradient-echo MRI showed delay of ischemic-hemorrhagic lesions in EPC-treated rats. Real-time RT-PCR pathway-specific array-analysis revealed age-associated gene expression changes in CD45-[CD34+/KDR]EPC subtypes, which were accelerated in stroke-prone rats. Pro-angiogenic genes implicated in intimal hyperplasia were increased in stroke-prone rat EPCs (P<0.0001), suggesting a maladaptive endothelial repair system which acts like a double-edged sword repairing while predisposing to age-associated intimal hyperplasia.Altogether, the data demonstrate that CD45-[CD34/KDR+]EPCs are juvenile protective factors for ischemic hemorrhagic stroke as modeled in the spTg25-rat model. The ability to delay stroke onset emphasizes the importance of EPC-mediated roles in vascular health for ischemic-hemorrhagic stroke, a high unmet need

Sex-specific hippocampus-dependent cognitive deficits and increased neuronal autophagy in DEspR haploinsufficiency in mice

Aside from abnormal angiogenesis, dual endothelin-1/VEGF signal peptide-activated receptor deficiency (DEspR−/−) results in aberrant neuroepithelium and neural tube differentiation, thus elucidating DEspR's role in neurogenesis. With the emerging importance of neurogenesis in adulthood, we tested the hypothesis that nonembryonic-lethal DEspR haploinsufficiency (DEspR+/−) perturbs neuronal homeostasis, thereby facilitating aging-associated neurodegeneration. Here we show that, in male mice only, DEspR-haploinsufficiency impaired hippocampus-dependent visuospatial and associative learning and induced noninflammatory spongiform changes, neuronal vacuolation, and loss in the hippocampus, cerebral cortex, and subcortical regions, consistent with autophagic cell death. In contrast, DEspR+/− females exhibited better cognitive performance than wild-type females and showed absence of neuropathological changes. Signaling pathway analysis revealed DEspR-mediated phosphorylation of activators of autophagy inhibitor mammalian target of rapamycin (mTOR) and dephosphorylation of known autophagy inducers. Altogether, the data demonstrate DEspR-mediated diametrical, sex-specific modulation of cognitive performance and autophagy, highlight cerebral neuronal vulnerability to autophagic dysregulation, and causally link DEspR haploinsufficiency with increased neuronal autophagy, spongiosis, and cognitive decline in mice

Aortic and Carotid Arterial Stiffness and Epigenetic Regulator Gene Expression Changes Precede Blood Pressure Rise in Stroke-Prone Dahl Salt-Sensitive Hypertensive Rats

<div><p>Multiple clinical studies show that arterial stiffness, measured as pulse wave velocity (PWV), precedes hypertension and is an independent predictor of hypertension end organ diseases including stroke, cardiovascular disease and chronic kidney disease. Risk factor studies for arterial stiffness implicate age, hypertension and sodium. However, causal mechanisms linking risk factor to arterial stiffness remain to be elucidated. Here, we studied the causal relationship of arterial stiffness and hypertension in the Na-induced, stroke-prone Dahl salt-sensitive (S) hypertensive rat model, and analyzed putative molecular mechanisms. Stroke-prone and non-stroke-prone male and female rats were studied at 3- and 6-weeks of age for arterial stiffness (PWV, strain), blood pressure, vessel wall histology, and gene expression changes. Studies showed that increased left carotid and aortic arterial stiffness preceded hypertension, pulse pressure widening, and structural wall changes at the 6-week time-point. Instead, differential gene induction was detected implicating molecular-functional changes in extracellular matrix (ECM) structural constituents, modifiers, cell adhesion, and matricellular proteins, as well as in endothelial function, apoptosis balance, and epigenetic regulators. Immunostaining testing histone modifiers Ep300, HDAC3, and PRMT5 levels confirmed carotid artery-upregulation in all three layers: endothelial, smooth muscle and adventitial cells. Our study recapitulates observations in humans that given salt-sensitivity, increased Na-intake induced arterial stiffness before hypertension, increased pulse pressure, and structural vessel wall changes. Differential gene expression changes associated with arterial stiffness suggest a molecular mechanism linking sodium to full-vessel wall response affecting gene-networks involved in vascular ECM structure-function, apoptosis balance, and epigenetic regulation.</p></div

Representative images for pulse wave velocity (PWV) and strain measurements.

<p><b>A</b>, Measurement of distance between two anatomical points along the abdominal aorta: proximal point after superior mesenteric artery branchpoint; distal point at site of crossing of renal vein. <b>B</b>, Representative Doppler frequency at distal point site: where renal vein crosses aorta. Integrated software for cursor-based measurement of distance given in mm (in <b>A</b>) and time in milliseconds from the peak of the ECG-R wave to the foot of the velocity upstroke (in <b>B</b>). <b>C</b>, Representative M-mode image for strain measurement in left carotid artery.</p

Development of high blood pressure in stoke-prone (SP) and non stroke-prone (nSP) Dahl S female rats.

<p>Systolic (<b>A</b>), diastolic (<b>B</b>), mean arterial (<b>C</b>) and pulse (<b>D</b>) pressures in SP Dahl S (n = 4, closed circles) and nSP Dahl S (n = 6, open circles) female rats. Blood pressure parameters were collected at 6 and 16 weeks of age. Values are means ± s.e.m. ***<i>P</i><0.001, (Two Way ANOVA followed by Holm-Sidak Test for multiple comparisons).</p

Comparative analysis of impact of age and stroke susceptibility on DEspR+ and DEspR- EPC gene expression profiles. (Specific details in Table S1).

<p>Graphical representation of significant gene-specific changes (fold-change: ratio test/common reference) comparing 2 m-old spTg25+ rat EPCs (spY) and non-stroke prone 4 m-old rat EPCs (nspO) reveals similar gene expression changes using 4 m-old spTg25+ rats (spO) as the common reference sample. In both DEspR+ and DEspR- EPCs, most of spY/spO gene expression changes (blue) are similar to nspO/spO gene expression changes (red) with few exceptions. More gene expression changes are noted in DEspR+ EPCs compared with DEspR- EPCs. Significance testing was obtained by 2-way [gene×EPC-subtype] ANOVA followed by Holm-Sidak test for multiple comparisons. Data [gene names, fold change, P-values] presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055222#pone.0055222.s001" target="_blank">Table S1</a>. Negative fold-change, decreased gene expression levels; positive fold-change, increased gene expression levels; zero level, non-expressed genes.</p