Renal ischemia alters the transcriptomic and epigenetic profile of inflammatory genes in swine scattered tubular-like cells.

BACKGROUND: Scattered tubular-like cells (STCs) are differentiated renal tubular cells that during recovery from ischemic injury dedifferentiate to repair other injured renal cells. Renal artery stenosis (RAS), often associated with chronic inflammatory injury, compromises the integrity and function of STCs, but the underlying mechanisms remain unknown. We hypothesized that RAS alters the transcriptomic and epigenetic profile of inflammatory genes in swine STCs. METHODS: STCs were harvested from pig kidneys after 10 weeks of RAS or sham (n=6 each). STC mRNA profiles of inflammatory genes were analyzed using high-throughput mRNA-sequencing (seq) and their DNA methylation (5mC) and hydroxymethylation (5hmC) profiles by DNA immunoprecipitation and next-generation sequencing (MeDIP-seq) (n=3 each), followed by an integrated (mRNA-seq/MeDIP-seq) analysis. STC protein expression of candidate differentially expressed (DE) genes and common proinflammatory proteins were subsequently assessed in vitro before and after epigenetic (Bobcat339) modulation. RESULTS: mRNA-seq identified 57 inflammatory genes up-regulated in RAS-STCs versus Normal-STCs (>1.4 or <0.7-fold, P<0.05), of which 14% exhibited lower 5mC and 5% higher 5hmC levels in RAS-STCs versus Normal-STCs, respectively. Inflammatory gene and protein expression was higher in RAS-STCs compared with Normal-STCs but normalized after epigenetic modulation. CONCLUSIONS: These observations highlight a novel modulatory mechanism of this renal endogenous repair system and support development of epigenetic or anti-inflammatory therapies to preserve the reparative capacity of STCs in individuals with RAS.

Human Obesity Attenuates Cardioprotection Conferred by Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells.

To explore the impact of obesity on reparative potency of adipose tissue-derived mesenchymal stromal/stem cells (A-MSC) in hypertensive cardiomyopathy, A-MSC were harvested from subcutaneous fat of obese and age-matched non-obese human subjects during bariatric or kidney donation surgeries, and then injected into mice 2 weeks after inducing renovascular hypertension (RVH) or sham surgery. Two weeks later, left ventricular (LV) function and deformation were estimated in vivo by micro-magnetic resonance imaging and myocardial damage ex vivo. Blood pressure and myocardial wall thickening were elevated in RVH + Vehicle and normalized only by lean-A-MSC. Both A-MSC types reduced LV mass and normalized the reduced LV peak strain radial in RVH, yet obese-A-MSC also impaired LV systolic function. A-MSC alleviated myocardial tissue damage in RVH, but lean-A-MSC decreased oxidative stress more effectively. Obese-A-MSC also showed increased cellular inflammation in vitro. Therefore, obese-A-MSC are less effective than lean-A-MSC in blunting hypertensive cardiomyopathy in mice with RVH.

Carotid Plaques From Symptomatic Patients Are Characterized by Local Increase in Xanthine Oxidase Expression.

Background and Purpose: XO (xanthine oxidase) is a key enzyme of uric acid metabolism and is thought to contribute to oxidative pathways that promote atherosclerotic plaque progression, yet its role in plaque destabilization is not well elucidated. We hypothesized that XO is expressed in carotid plaque from symptomatic patients in association with cardiovascular risk factors. Methods: Patients were stratified by symptoms, defined as presentation with an ipsilateral cerebral ischemic event. Carotid atherosclerotic plaques were obtained from 44 patients with symptomatic plaque and 44 patients without ischemic cerebral events. Protein expression of XO was evaluated by immunohistochemical staining and the percentage of cells expressing XO and CD68 (macrophage marker) compared between the groups. Biochemical and demographic cardiometabolic risk factors of study participants also were measured. Results: Carotid atherosclerotic plaques from symptomatic patients were associated with significantly higher XO expression versus asymptomatic plaque (median [interquartile range]: 1.24 [2.09] versus 0.16 [0.34]; P<0.001) and with significantly higher circulating uric acid levels (mean±SD: 7.36±2.10 versus 5.37±1.79 mg/dL; P<0.001, respectively). In addition, XO expression in atherosclerotic carotid plaque was inversely associated with serum high-density lipoproteins cholesterol levels (P=0.010, r=−0.30) and directly with circulating uric acid levels (P<0.001, r=0.45). The average percentage of macrophages that expressed XO was significantly higher in symptomatic versus asymptomatic plaques (median [interquartile range]: 93.37% [25] versus 46.15% [21], respectively; P<0.001). Conclusions: XO overexpression in macrophages is associated with increased serum uric acid and low high-density lipoproteins cholesterol levels and may potentially have a mechanistic role in carotid plaque destabilization. The current study supports a potential role for uric acid synthesis pathway as a target for management of carotid atherosclerosis in humans.

Hypoxic preconditioning induces epigenetic changes and modifies swine mesenchymal stem cell angiogenesis and senescence in experimental atherosclerotic renal artery stenosis.

BACKGROUND: Atherosclerotic renal artery stenosis (ARAS) is a risk factor for ischemic and hypertensive kidney disease (HKD) for which autologous mesenchymal stem cell (MSC) appears to be a promising therapy. However, MSCs from ARAS patients exhibit impaired function, senescence, and DNA damage, possibly due to epigenetic mechanisms. Hypoxia preconditioning (HPC) exerts beneficial effects on cellular proliferation, differentiation, and gene and protein expression. We hypothesized that HPC could influence MSC function and senescence in ARAS by epigenetic mechanisms and modulating gene expression of chromatin-modifying enzymes. METHODS: Adipose-derived MSC harvested from healthy control (N = 8) and ARAS (N = 8) pigs were cultured under normoxia (20%O2) or hypoxia (1%O2) conditions. MSC function was assessed by migration, proliferation, and cytokine release in conditioned media. MSC senescence was evaluated by SA-ß-gal activity. Specific pro-angiogenic and senescence genes were assessed by reverse transcription polymerase chain reaction (RT-PCR). Dot blotting was used to measure global genome 5-hydroxymethylcytosine (5hmC) levels on DNA and Western blotting of modified histone 3 (H3) proteins to quantify tri-methylated lysine-4 (H3K4me3), lysine-9 (H3K9me3), and lysine-27 (H3K27me3) residues. RESULTS: Specific pro-angiogenic genes in ARAS assessed by RT-PCR were lower at baseline but increased under HPC, while pro-senescence genes were higher in ARAS at baseline as compared healthy MSCs. ARAS MSCs under basal conditions, displayed higher H3K4me3, H3K27me3, and 5hmC levels compared to healthy MSCs. During HPC, global 5hmC levels were decreased while no appreciable changes occurred in histone H3 tri-methylation. ARAS MSCs cultured under HPC had higher migratory and proliferative capacity as well as increased vascular endothelial growth factor and epidermal growth factor expression compared to normoxia, and SA-ß-gal activity decreased in both animal groups. CONCLUSIONS: These data demonstrate that swine ARAS MSCs have decreased angiogenesis and increased senescence compared to healthy MSCs and that HPC mitigates MSC dysfunction, senescence, and DNA hydroxymethylation in ARAS MSC. Thus, HPC for MSCs may be considered for their optimization to improve autologous cell therapy in patients with nephropathies.

Quercetin Reverses Cardiac Systolic Dysfunction in Mice Fed with a High-Fat Diet: Role of Angiogenesis.

Global consumption of high-fat diets (HFD) is associated with an increased incidence of cardiometabolic syndrome and cardiac injury, warranting identification of cardioprotective strategies. Cardioprotective effects of quercetin (Q) have mostly been evaluated in ischemic heart disease models and attributed to senolysis. We hypothesized that Q could alleviate murine cardiac damage caused by HFD by restoring the myocardial microcirculation. C57BL/6J mice were fed standard chow or HFD for 6 months and then treated with Q (50 mg/kg) or vehicle 5-day biweekly for 10 additional weeks. Left ventricular (LV) cardiac function was studied in vivo using magnetic resonance imaging, and intramyocardial fat deposition, microvascular density, oxidative stress, and senescence were analyzed ex vivo. Additionally, direct angiogenic effects of Q were studied in vitro in HUVECs. HFD increased body weight, heart weight, total cholesterol, and triglyceride levels, whereas Q normalized heart weight and triglycerides. LV ejection fraction was lower in HFD vs. control mice (56.20 ± 15.8% vs. 73.38 ± 5.04%, respectively, P < 0.05), but improved in HFD + Q mice (67.42 ± 7.50%, P < 0.05, vs. HFD). Q also prevented cardiac fat accumulation and reduced HFD-induced cardiac fibrosis, cardiomyocyte hypertrophy, oxidative stress, and vascular rarefaction. Cardiac senescence was not observed in any group. In vitro, ox-LDL reduced HUVEC tube formation activity, which Q effectively improved. Quercetin may directly induce angiogenesis and decrease myocardial oxidative stress, which might account for its cardioprotective effects in the murine HFD-fed murine heart independently from senolytic activity. Furthermore, its beneficial effects might be partly attributed to a decrease in plasma triglycerides and intramyocardial fat deposition.

Endovascular reversal of renovascular hypertension blunts cardiac dysfunction and deformation in swine.

OBJECTIVE: Renovascular hypertension (RVH) induces hemodynamic and humoral aberrations that may impair cardiac function, structure and mechanics, including cardiac twist and deformation. Revascularization of a stenotic renal artery can decrease blood pressure (BP), but its ability to restore cardiac mechanics in RVH remains unclear. We hypothesized that percutaneous transluminal renal angioplasty (PTRA) would improve cardiac function and left ventricular (LV) deformation in swine RVH. METHODS: Seventeen domestic pigs were studied for 16 weeks: RVH, RVH + PTRA and normal controls (n = 5-6 each). Global LV function was estimated by multidetector computed-tomography, and LV deformation by electrocardiographically triggered MRI tagging at the apical, mid, and basal LV levels. Cardiomyocyte hypertrophy, myocardial capillary density, and fibrosis were evaluated ex vivo. RESULTS: BP and wall thickness were elevated in RVH and decreased by PTRA, yet remained higher than in controls. LV myocardial muscle mass increased in RVH pigs, which also developed diastolic dysfunction, whereas cardiac output increased. Furthermore, both apical rotation and peak torsion angle increased in RVH compared with controls. Ex vivo, RVH induced myocardial fibrosis and vascular rarefaction. PTRA restored cardiac function and alleviated hypertrophy, vascular rarefaction, and fibrosis. PTRA also normalized apical rotation and peak torsion angle, and elevated basal peak radial strain and apical peak radial strain compared with RVH. CONCLUSION: In addition to cardiac LV adaptive hypertrophy and diastolic dysfunction, short-term RVH causes cardiac deformation. Despite only partial improvement in BP, PTRA effectively restored cardiac function and reversed abnormal mechanics. Hence, renal revascularization may be a useful strategy to preserve cardiac function in RVH.

Targeting senescence improves angiogenic potential of adipose-derived mesenchymal stem cells in patients with preeclampsia.

BACKGROUND: Preeclampsia is a pregnancy-specific hypertensive disorder characterized by impaired angiogenesis. We postulate that senescence of mesenchymal stem cells (MSC), multipotent cells with pro-angiogenic activities, is one of the mechanisms by which systemic inflammation exerts inhibitory effects on angiogenesis in preeclampsia. METHODS: MSC were isolated from abdominal fat tissue explants removed during medically indicated C-sections from women with preeclampsia (PE-MSC, n = 10) and those with normotensive pregnancies (NP-MSC, n = 12). Sections of the frozen subcutaneous adipose tissue were assessed for inflammation by staining for tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein (MCP)-1. Viability, proliferation, and migration were compared between PE-MSC vs. NP-MSC. Apoptosis and angiogenesis were assayed before and after treatment with a senolytic agent (1 µM dasatinib) using the IncuCyte S3 Live-Cell Analysis System. Similarly, staining for senescence-associated beta galactosidase (SABG) and qPCR for gene expression of senescence markers, p16 and p21, as well as senescence-associated secretory phenotype (SASP) components, IL-6, IL-8, MCP-1, and PAI-1, were studied before and after treatment with dasatinib and compared between PE and NP. RESULTS: After in vitro exposure to TNF-alpha, MSC demonstrated upregulation of SASP components, including interleukins-6 and -8 and MCP-1. Staining of the subcutaneous adipose tissue sections revealed a greater inflammatory response in preeclampsia, based on the higher levels of both TNF-alpha and MCP-1 compared to normotensive pregnancies (p < 0.001 and 0.024, respectively). MSC isolated from PE demonstrated a lower percentage of live MSC cells (p = 0.012), lower proliferation (p = 0.005), and higher migration (p = 0.023). At baseline, PE-MSC demonstrated a senescent phenotype, reflected by more abundant staining for SABG (p < 0.001), upregulation of senescence markers and SASP components, as well as lower angiogenic potential (p < 0.001), compared to NP-MSC. Treatment with dasatinib increased significantly the number of apoptotic PE-MSC compared to NP-MSC (0.011 vs. 0.093) and decreased the gene expression of p16 and six SASP components. The mechanistic link between senescence and impaired angiogenesis in PE was confirmed by improved angiogenic potential of PE-MSC (p < 0.001) after dasatinib treatment. CONCLUSIONS: Our data suggest that MSC senescence exerts inhibitory effects on angiogenesis in preeclampsia. Senolytic agents may offer the opportunity for mechanism-based therapies.

Characterization of microbial communities in gas industry pipelines.

Culture-independent techniques, denaturing gradient gel electrophoresis (DGGE) analysis, and random cloning of 16S rRNA gene sequences amplified from community DNA were used to determine the diversity of microbial communities in gas industry pipelines. Samples obtained from natural gas pipelines were used directly for DNA extraction, inoculated into sulfate-reducing bacterium medium, or used to inoculate a reactor that simulated a natural gas pipeline environment. The variable V2-V3 (average size, 384 bp) and V3-V6 (average size, 648 bp) regions of bacterial and archaeal 16S rRNA genes, respectively, were amplified from genomic DNA isolated from nine natural gas pipeline samples and analyzed. A total of 106 bacterial 16S rDNA sequences were derived from DGGE bands, and these formed three major clusters: beta and gamma subdivisions of Proteobacteria and gram-positive bacteria. The most frequently encountered bacterial species was Comamonas denitrificans, which was not previously reported to be associated with microbial communities found in gas pipelines or with microbially influenced corrosion. The 31 archaeal 16S rDNA sequences obtained in this study were all related to those of methanogens and phylogenetically fall into three clusters: order I, Methanobacteriales; order III, Methanomicrobiales; and order IV, Methanosarcinales: Further microbial ecology studies are needed to better understand the relationship among bacterial and archaeal groups and the involvement of these groups in the process of microbially influenced corrosion in order to develop improved ways of monitoring and controlling microbially influenced corrosion.

16S rRNA-based analysis of microbiota from the cecum of broiler chickens.

The microbiota of the intestinal tract of chickens plays an important role in inhibiting the establishment of intestinal pathogens. Earlier culturing and microscopic examinations indicated that only a fraction of the bacteria in the cecum of chickens could be grown in the laboratory. Therefore, a survey of cecal bacteria was done by retrieval of 16S rRNA gene sequences from DNA isolated from the cecal content and the cecal mucosa. The ribosomal gene sequences were amplified with universal primers and cloned or subjected to temporal temperature gradient gel electrophoresis (TTGE). Partial 16S rRNA gene sequences were determined from the clones and from the major bands in TTGE gels. A total of 1,656 partial 16S rRNA gene sequences were obtained and compared to sequences in the GenBank. The comparison indicated that 243 different sequences were present in the samples. Overall, sequences representing 50 phylogenetic groups or subgroups of bacteria were found, but approximately 89% of the sequences represented just four phylogenetic groups (Clostridium leptum, Sporomusa sp., Clostridium coccoides, and enterics). Sequences of members of the Bacteroides group, the Bifidobacterium infantis subgroup, and of Pseudomonas sp. each accounted for less than 2% of the total. Sequences related to those from the Escherichia sp. subgroup and from Lactobacillus, Pseudomonas, and Bifidobacterium spp. were generally between 98 and 100% identical to sequences already deposited in the GenBank. Sequences most closely related to those of the other bacteria were generally 97% or less identical to those in the databases and therefore might be from currently unknown species. TTGE and random cloning indicated that certain phylogenetic subgroups were common to all birds analyzed, but sequence data from random cloning also provided evidence for qualitative and quantitative differences among the cecal microbiota of individual birds reared under very similar conditions.