Kv2.1 channels prevent vasomotion and safeguard myogenic reactivity in rat small superior cerebellar arteries

Vascular smooth muscle voltage-gated potassium (Kv) channels have been proposed to contribute to myogenic autoregulation. Surprisingly, in initial experiments, we observed that the Kv2 channel inhibitor stromatoxin induced vasomotion without affecting myogenic tone. Thus, we tested the hypothesis that Kv2 channels contribute to myogenic autoregulation by fine-tuning the myogenic response. Expression of Kv2 channel mRNA was determined using real-time PCR and ‘multiplex’ single-cell RT-PCR. Potassium currents were measured using the patch-clamp technique. Contractile responses of intact arteries were studied using isobaric myography. Expression of Kv2.1 but not Kv2.2 channels was detected in intact rat superior cerebellar arteries and in single smooth muscle cells. Stromatoxin, a high-affinity inhibitor of Kv2 channels, reduced smooth muscle Kv currents by 61% at saturating concentrations (EC50 36 nmol/L). Further, stromatoxin (10–100 nmol/L) induced pronounced vasomotion in 48% of the vessels studied. In vessels not exhibiting vasomotion, stromatoxin did not affect myogenic reactivity. Notably, in vessels exhibiting stromatoxin-induced vasomotion, pressure increases evoked two effects: First, they facilitated the occurrence of random vasodilations and/or vasoconstrictions, disturbing the myogenic response (24% of the vessels). Second, they modified the vasomotion by decreasing its amplitude and increasing its frequency, thereby destabilizing myogenic tone (76% of the vessels). Our study demonstrates that (i) Kv2.1 channels are the predominantly expressed Kv channels in smooth muscle cells of rat superior cerebellar arteries, and (ii) Kv2.1 channels provide a novel type of negative feedback mechanism in myogenic autoregulation by preventing vasomotion and thereby safeguarding the myogenic response

Comparative Analysis of Podocyte Foot Process Morphology in Three Species by 3D Super-Resolution Microscopy

Since the size selectivity of the filtration barrier and kidney function are highly dependent on podocyte foot process morphology, visualization of foot processes is important. However, the size of foot processes is below the optical resolution of light microscopy. Therefore, electron microcopy has been indispensable to detect changes in foot process morphology so far, but it is a sophisticated and time-consuming technique. Recently, our group has shown that 3D structured illumination microscopy (3D-SIM), a super-resolution microscopy (SRM) technique, can visualize individual foot processes in human biopsies. Moreover, we have developed a software-based approach to directly quantify the structure of podocyte foot processes named Podocyte Exact Morphology Measurement Procedure (PEMP). As shown in patients suffering from minimal change disease (MCD), PEMP allows the quantification of changes of the foot process morphology by measuring the filtration slit density (FSD). Since rodents are frequently used in basic research, we have applied PEMP to quantify foot processes of mice and rats. Comparative analysis of nephrin-stained kidneys from humans, rats, and mice showed significant differences of the FSD. The highest FSD was measured in mice (3.83 ± 0.37 μm−1; mean ± SD) followed by rats (3.36 ± 0.42 μm−1) and humans (3.11 ± 0.26 μm−1). To demonstrate that PEMP can be used to determine foot process morphology also in affected animals, we measured the FSD in palladin-knockout mice on a 129S1 genetic background compared to wild-type littermates. Taken together, we established a method for the quick and exact quantification of podocyte foot process morphology which can be applied to diagnosis and basic research

Angiotensin receptor-neprilysin inhibitor improves coronary collateral perfusion

BACKGROUND: We investigated the pleiotropic effects of an angiotensin receptor-neprilysin inhibitor (ARNi) on collateral-dependent myocardial perfusion in a rat model of coronary arteriogenesis, and performed comprehensive analyses to uncover the underlying molecular mechanisms. METHODS: A rat model of coronary arteriogenesis was established by implanting an inflatable occluder on the left anterior descending coronary artery followed by a 7-day repetitive occlusion procedure (ROP). Coronary collateral perfusion was measured by using a myocardial particle infusion technique. The putative ARNi-induced pro-arteriogenic effects were further investigated and compared with an angiotensin-converting enzyme inhibitor (ACEi). Expression of the membrane receptors and key enzymes in the natriuretic peptide system (NPS), renin-angiotensin-aldosterone system (RAAS) and kallikrein-kinin system (KKS) were analyzed by quantitative polymerase chain reaction (qPCR) and immunoblot assay, respectively. Protein levels of pro-arteriogenic cytokines were measured by enzyme-linked immunosorbent assay, and mitochondrial DNA copy number was assessed by qPCR due to their roles in arteriogenesis. Furthermore, murine heart endothelial cells (MHEC5-T) were treated with a neprilysin inhibitor (NEPi) alone, or in combination with bradykinin receptor antagonists. MHEC5-T proliferation was analyzed by colorimetric assay. RESULTS: The in vivo study showed that ARNis markedly improved coronary collateral perfusion, regulated the gene expression of KKS, and increased the concentrations of relevant pro-arteriogenic cytokines. The in vitro study demonstrated that NEPis significantly promoted MHEC5-T proliferation, which was diminished by bradykinin receptor antagonists. CONCLUSION: ARNis improve coronary collateral perfusion and exert pro-arteriogenic effects via the bradykinin receptor signaling pathway

Baroreflex sensitivity differs among same strain Wistar rats from the same laboratory

Previous studies showed that a proportion of normotensive Sprague-Dawley rats spontaneously exhibit lower baroreflex sensitivity. However, investigations have not yet been carried out on Wistar rats. We aimed to compare baroreflex sensitivity among rats from the same strain and the same laboratory. Male Wistar normotensive rats (300–400g) were studied. Cannulas were inserted into the abdominal aortic artery through the right femoral artery to measure mean arterial pressure and heart rate. Baroreflex was calculated as the derivative of the variation of heart rate in function of the mean arterial pressure variation (ΔHR/ΔMAP) tested with a depressor dose of sodium nitroprusside (50 µg/kg) and with a pressor dose of phenylephrine (8µg/kg) in the right femoral venous approach through an inserted cannula. We divided the rats into four groups: i) high bradycardic baroreflex, baroreflex gain less than −2 tested with phenylephrine; ii) low bradycardic baroreflex, baroreflex gain between −1 and −2 tested with phenylephrine; iii) high tachycardic baroreflex, baroreflex gain less than −3 tested with sodium nitroprusside; and iv) low tachycardic baroreflex, baroreflex gain between −1 and −3 tested with sodium nitroprusside. Approximately 71% of the rats presented a decrease in bradycardic reflex while around half showed an increase in tachycardic reflex. No significant changes in basal mean arterial pressure and heart rate, tachycardic and bradycardic peak and heart rate range were observed. There was a significant change in baroreflex sensitivity among rats from the same strain and the same laboratory

Nicotinic acetylcholine receptor subunit variants are associated with blood pressure; findings in the Old Order Amish and replication in the Framingham Heart Study

<p>Abstract</p> <p>Background</p> <p>Systemic blood pressure, influenced by both genetic and environmental factors, is regulated via sympathetic nerve activity. We assessed the role of genetic variation in three subunits of the neuromuscular nicotinic acetylcholine receptor positioned on chromosome 2q, a region showing replicated evidence of linkage to blood pressure.</p> <p>Methods</p> <p>We sequenced <it>CHRNA1</it>, <it>CHRND </it>and <it>CHRNG </it>in 24 Amish subjects from the Amish Family Diabetes Study (AFDS) and identified 20 variants. We then performed association analysis of non-redundant variants (n = 12) in the complete AFDS cohort of 1,189 individuals, and followed by genotyping blood pressure-associated variants (n = 5) in a replication sample of 1,759 individuals from the Framingham Heart Study (FHS).</p> <p>Results</p> <p>The minor allele of a synonymous coding SNP, rs2099489 in <it>CHRNG</it>, was associated with higher systolic blood pressure in both the Amish (p = 0.0009) and FHS populations (p = 0.009) (minor allele frequency = 0.20 in both populations).</p> <p>Conclusion</p> <p><it>CHRNG </it>is currently thought to be expressed only during fetal development. These findings support the Barker hypothesis, that fetal genotype and intra-uterine environment influence susceptibility to chronic diseases later in life. Additional studies of this variant in other populations, as well as the effect of this variant on acetylcholine receptor expression and function, are needed to further elucidate its potential role in the regulation of blood pressure. This study suggests for the first time in humans, a possible role for genetic variation in the neuromuscular nicotinic acetylcholine receptor, particularly the gamma subunit, in systolic blood pressure regulation.</p

Sprouted Innervation into Uterine Transplants Contributes to the Development of Hyperalgesia in a Rat Model of Endometriosis

Endometriosis is an enigmatic painful disorder whose pain symptoms remain difficult to alleviate in large part because the disorder is defined by extrauteral endometrial growths whose contribution to pain is poorly understood. A rat model (ENDO) involves autotransplanting on abdominal arteries uterine segments that grow into vascularized cysts that become innervated with sensory and sympathetic fibers. ENDO rats exhibit vaginal hyperalgesia. We used behavioral, physiological, and immunohistochemical methods to test the hypothesis that cyst innervation contributes to the development of this hyperalgesia after transplant. Rudimentary sensory and sympathetic innervation appeared in the cysts at two weeks, sprouted further and more densely into the cyst wall by four weeks, and matured by six weeks post-transplant. Sensory fibers became abnormally functionally active between two and three weeks post-transplant, remaining active thereafter. Vaginal hyperalgesia became significant between four and five weeks post-transplant, and stabilized after six to eight weeks. Removing cysts before they acquired functional innervation prevented vaginal hyperalgesia from developing, whereas sham cyst removal did not. Thus, abnormally-active innervation of ectopic growths occurs before hyperalgesia develops, supporting the hypothesis. These findings suggest that painful endometriosis can be classified as a mixed inflammatory/neuropathic pain condition, which opens new avenues for pain relief. The findings also have implications beyond endometriosis by suggesting that functionality of any transplanted tissue can be influenced by the innervation it acquires

Leukocyte telomere length and mitochondrial DNA copy number associate with endothelial function in aging-related cardiovascular disease

BackgroundWe investigated the association between leukocyte telomere length, mitochondrial DNA copy number, and endothelial function in patients with aging-related cardiovascular disease (CVD).MethodsIn total 430 patients with CVD and healthy persons were enrolled in the current study. Peripheral blood was drawn by routine venipuncture procedure. Plasma and peripheral blood mononuclear cells (PBMCs) were collected. Cell-free genomic DNA (cfDNA) and leukocytic genomic DNA (leuDNA) were extracted from plasma and PBMCs, respectively. Relative telomere length (TL) and mitochondrial DNA copy number (mtDNA-CN) were analyzed using quantitative polymerase chain reaction. Endothelial function was evaluated by measuring flow-mediated dilation (FMD). The correlation between TL of cfDNA (cf-TL), mtDNA-CN of cfDNA (cf-mtDNA), TL of leuDNA (leu-TL), mtDNA-CN of leuDNA (leu-mtDNA), age, and FMD were analyzed based on Spearman's rank correlation. The association between cf-TL, cf-mtDNA, leu-TL, leu-mtDNA, age, gender, and FMD were explored using multiple linear regression analysis.Resultscf-TL positively correlated with cf-mtDNA (r = 0.1834, P = 0.0273), and leu-TL positively correlated with leu-mtDNA (r = 0.1244, P = 0.0109). In addition, both leu-TL (r = 0.1489, P = 0.0022) and leu-mtDNA (r = 0.1929, P &lt; 0.0001) positively correlated with FMD. In a multiple linear regression analysis model, both leu-TL (β = 0.229, P = 0.002) and leu-mtDNA (β = 0.198, P = 0.008) were positively associated with FMD. In contrast, age was inversely associated with FMD (β = −0.426, P &lt; 0.0001).ConclusionTL positively correlates mtDNA-CN in both cfDNA and leuDNA. leu-TL and leu-mtDNA can be regarded as novel biomarkers of endothelial dysfunction

Consequences of perinatal treatment with l-arginine and antioxidants for the renal transcriptome in spontaneously hypertensive rats

Treating spontaneously hypertensive rats (SHR) with l-arginine, taurine, and vitamins C and E (ATCE) during nephrogenesis (2 weeks before to 4 weeks after birth) persistently lowers blood pressure. Hypothetically, differential gene expression in kidney of SHR vs. normotensive Wistar–Kyoto rats (WKY) is partially corrected by maternal ATCE in SHR. Differential gene expression in 2-days, 2-weeks, and 48-week-old rats was studied using oligonucleotide chips. Transcription factor binding sites (TFBS) of differentially expressed genes were analyzed in silico. Differential gene expression varied between SHR+ATCE and SHR, suggesting both direct and indirect effects; but, few genes were modulated toward WKY level and there was little overlap between ages. TFBS analysis suggests less Elk-1-driven gene transcription in both WKY and SHR+ATCE vs. SHR at 2 days and 2 weeks. Concluding, in SHR, persistent antihypertensive effects of maternal ATCE are not primarily due to persistent corrective transcription. Less Elk-1-driven transcription at 2 days and 2 weeks may be involved

CD43Lo classical monocytes participate in the cellular immune response to isolated primary blast lung injury

BACKGROUNDUnderstanding of the cellular immune response to primary blast lung injury (PBLI) is limited, with only the neutrophil response well documented. Moreover, its impact on the immune response in distal organs remains poorly understood. In this study, a rodent model of isolated primary blast injury was used to investigate the acute cellular immune response to isolated PBLI in the circulation and lung, including the monocyte response, and investigate distal subacute immune effects in the spleen and liver 6 hours after injury.METHODSRats were subjected to a shock wave (~135 kPa overpressure, 2 ms duration) inducing PBLI or sham procedure. Rat physiology was monitored, and at 1, 3, and 6 hours thereafter, blood, lung, and bronchoalveolar lavage fluid (BALF) were collected and analyzed by flow cytometry, enzyme-linked immunosorbent assay, and histologic examination. In addition, at 6 hours, spleen and liver were collected and analyzed by flow cytometry.RESULTSLung histology confirmed pulmonary barotrauma and inflammation. This was associated with rises in CXCL-1, interleukin 6 (IL-6), tumor necrosis factor α and albumin protein in the BALF. Significant acute increases in blood and lung neutrophils and CD43Lo/His48Hi (classical) monocytes/macrophages were detected. No significant changes were seen in blood or lung “nonclassical” monocyte and in natural killler, B, or T cells. In the BALF, significant increases were seen in neutrophils, CD43Lo monocyte-macrophages and monocyte chemoattractant protein-1. Significant increases in CD43Lo and Hi monocyte-macrophages were detected in the spleen at 6 hours.CONCLUSIONThis study reveals a robust and selective response of CD43Lo/His48Hi (classical) monocytes, in addition to neutrophils, in blood and lung tissue following PBLI. An increase in monocyte-macrophages was also observed in the spleen at 6 hours. This profile of immune cells in the blood and BALF could present a new research tool for translational studies seeking to monitor, assess, or attenuate the immune response in blast-injured patients