Ning Hou

Hypersensitivity of BKCa to Ca2+ Sparks Underlies Hyporeactivity of Arterial Smooth Muscle in Shock

Large conductance Ca2+-activated K+ channels (BKCa) play a critical role in blood pressure regulation by tuning the vascular smooth muscle tone, and hyposensitivity of BKCa to Ca2+ sparks resulting from its altered &bgr;1 subunit stoichiometry underlies vasoconstriction in animal models of hypertension. Here we demonstrate hypersensitivity of BKCa to Ca2+ sparks that contributes to hypotension and blunted vasoreactivity in acute hemorrhagic shock. In arterial smooth muscle cells under voltage-clamp conditions (0 mV), the amplitude and duration, but not the frequency, of spontaneous transient outward currents of BKCa origin were markedly enhanced in hemorrhagic shock, resulting in a 265% greater hyperpolarizing current. Concomitantly, subsurface Ca2+ spark frequency was either unaltered (at 0 mV) or decreased in hyperpolarized resting cells. Examining the relationship between spark and spontaneous transient outward current amplitudes revealed a hypersensitive BKCa activity to Ca2+ spark in hemorrhagic shock, whereas the spark–spontaneous transient outward current coupling fidelity was near unity in both groups. Importantly, we found an acute upregulation of the &bgr;1 subunit of the channel, and single-channel recording substantiated BKCa hypersensitivity at micromolar Ca2+, which promotes the &agr; and &bgr;1 subunit interaction. Treatment of shock animals with the BKCa inhibitors iberiotoxin and charybdotoxin partially restored vascular membrane potential and vasoreactivity to norepinephrine and blood reinfusion. Thus, the results underscore a dynamic regulation of the BKCa–Ca2+ spark coupling and its therapeutic potential in hemorrhagic shock–associated vascular disorders.

Rhesus Macaques Develop Metabolic Syndrome With Reversible Vascular Dysfunction Responsive to Pioglitazone

Background— The metabolic syndrome (MetS) is a constellation of clinical features that include central obesity, hypertension, atherogenic dyslipidemia, and insulin resistance. However, the concept remains controversial; it has been debated whether MetS represents nothing more than simultaneous co-occurrence of individual risk factors or whether there are common shared pathophysiological mechanisms that link the individual components. Methods and Results— To investigate the emergence of metabolic and cardiovascular components during the development of MetS, we identified MetS-predisposed animals (n=35) in a large population of rhesus macaques (Macaca mulatta, 12.7±2.9 years old, n=408), acclimated them to standardized conditions, and monitored the progression of individual component features over 18 months. In 18 MetS animals with recently developed fasting hyperinsulinemia, central obesity, hypertension, and atherogenic dyslipidemia, we found that individual metabolic and cardiovascular components track together during the transition from pre-MetS to onset of MetS; MetS was associated with a 60% impairment of flow-mediated dilation, establishing the mechanistic link with vascular dysfunction. Pioglitazone treatment (3 mg/kg body weight/d for 6 weeks), a peroxisome proliferator–activated receptor &ggr; agonist, reversibly improved atherogenic dyslipidemia and insulin resistance and fully restored flow-mediated dilation with persistent benefits. Conclusions— Coemergence of metabolic and cardiovascular components during MetS progression and complete normalization of vascular dysfunction with peroxisome proliferator-activated receptor &ggr; agonists suggest shared underlying mechanisms rather than separate processes, arguing for the benefit of early intervention of MetS components. Predictive nonhuman primate (NHP) models of MetS should be highly valuable in mechanistic and translational studies on the pathogenesis of MetS in relation to cardiovascular disease and diabetes mellitus.

Overnutrition stimulates intestinal epithelium proliferation through β-catenin signaling in obese mice

Obesity is a major risk factor for type 2 diabetes and cardiovascular diseases. And overnutrition is a leading cause of obesity. After most nutrients are ingested, they are absorbed in the small intestine. Signals from β-catenin are essential to maintain development of the small intestine and homeostasis. In this study, we used a hyperphagia db/db obese mouse model and a high-fat diet (HFD)-induced obesity mouse model to investigate the effects of overnutrition on intestinal function and β-catenin signaling. The β-catenin protein was upregulated along with inactivation of glycogen synthase kinase (GSK)-3β in the intestines of both db/db and HFD mice. Proliferation of intestinal epithelial stem cells, villi length, nutrient absorption, and body weight also increased in both models. These changes were reversed by caloric restriction in db/db mice and by β-catenin inhibitor JW55 (a small molecule that increases β-catenin degradation) in HFD mice. Parallel, in vitro experiments showed that β-catenin accumulation and cell proliferation stimulated by glucose were blocked by the β-catenin inhibitor FH535. And the GSK-3 inhibitor CHIR98014 in an intestinal epithelial cell line increased β-catenin accumulation and cyclin D1 expression. These results suggested that, besides contribution to intestinal development and homeostasis, GSK-3β/β-catenin signaling plays a central role in intestinal morphological and functional changes in response to overnutrition. Manipulating the GSK-3β/β-catenin signaling pathway in intestinal epithelium might become a therapeutic intervention for obesity induced by overnutrition.

RhesusBase: a knowledgebase for the monkey research community

Although the rhesus macaque is a unique model for the translational study of human diseases, currently its use in biomedical research is still in its infant stage due to error-prone gene structures and limited annotations. Here, we present RhesusBase for the monkey research community (http://www.rhesusbase.org). We performed strand-specific RNA-Seq studies in 10 macaque tissues and generated 1.2 billion 90-bp paired-end reads, covering >97.4% of the putative exon in macaque transcripts annotated by Ensembl. We found that at least 28.7% of the macaque transcripts were previously mis-annotated, mainly due to incorrect exon–intron boundaries, incomplete untranslated regions (UTRs) and missed exons. Compared with the previous gene models, the revised transcripts show clearer sequence motifs near splicing junctions and the end of UTRs, as well as cleaner patterns of exon–intron distribution for expression tags and cross-species conservation scores. Strikingly, 1292 exon–intron boundary revisions between coding exons corrected the previously mis-annotated open reading frames. The revised gene models were experimentally verified in randomly selected cases. We further integrated functional genomics annotations from >60 categories of public and in-house resources and developed an online accessible database. User-friendly interfaces were developed to update, retrieve, visualize and download the RhesusBase meta-data, providing a ‘one-stop’ resource for the monkey research community.

Spontaneous periodontitis is associated with metabolic syndrome in rhesus monkeys

OBJECTIVE The present study was designed to investigate (1) whether the non-human primate would be an appropriate animal model for the study of spontaneous periodontitis and its association with metabolic syndrome (MetS), and (2) whether microRNAs (miRNAs) play roles in the co-development of metabolic disorders and periodontitis. DESIGN Rhesus monkeys (aged 12-29 years) with or without MetS were analyzed for the prevalence of periodontitis. The potential mechanisms underlying the association between MetS and periodontitis were explored using miRNA profiling of the gingival tissues from the MetS monkey groups with or without periodontitis as well as the age-matched controls. RESULTS Among the 57 rhesus monkeys examined, 18 were diagnosed with periodontitis according to the inclusion criteria, with an overall prevalence of 31.6%. Moreover, the prevalence of periodontitis was 8.3% in the control group, 18.2% in the at-risk group, and 44.1% in the MetS group. The C-reactive protein level was doubled in the MetS periodontitis group, compared to the non-periodontitis sub-groups. Most importantly, only 3 miRNAs were confirmed to be differentially expressed between the MetS periodontitis and non-periodontitis subgroups while other miRNAs showed similar expression profiles. CONCLUSIONS The results indicate that the monkey with MetS is an ideal model for studies of spontaneous periodontitis and its association with MetS. miRNA profiling using this unique model showed that miRNAs play roles in the co-development of MetS and periodontitis.

RIPK1 Binds MCU to Mediate Induction of Mitochondrial Ca2+ Uptake and Promotes Colorectal Oncogenesis

The receptor-interacting protein kinase 1 (RIPK1) is an essential signaling molecule in pathways for cell survival, apoptosis, and necroptosis. We report here that RIPK1 is upregulated in human colorectal cancer and promotes cell proliferation when overexpressed in a colon cancer cell line. RIPK1 interacts with mitochondrial Ca2+ uniporter (MCU) to promote proliferation by increasing mitochondrial Ca2+ uptake and energy metabolism. The ubiquitination site of RIPK1 (RIPK1-K377) was critical for this interaction with MCU and function in promoting cell proliferation. These findings identify the RIPK1-MCU pathway as a promising target to treat colorectal cancer.Significance: RIPK1-mediated cell proliferation through MCU is a central mechanism underlying colorectal cancer progression and may prove to be an important therapeutic target for colorectal cancer treatment. Cancer Res; 78(11); 2876-85. ©2018 AACR.

Eplerenone restores 24-h blood pressure circadian rhythm and reduces advanced glycation end-products in rhesus macaques with spontaneous hypertensive metabolic syndrome.

Hypertension is often associated with metabolic syndrome (MetS), and serves as a risk factor of MetS and its complications. Blood pressure circadian rhythm in hypertensive patients has been suggested to contribute to cardiovascular consequences and organ damage of hypertension. But circadian changes of BP and their response to drugs have not been clearly investigated in non-human primates (NHPs) of MetS with hypertension. Here, we identified 16 elderly, hypertensive MetS rhesus monkeys from our in-house cohort. With implanted telemetry, we investigate BP changes and its circadian rhythm, together with the effect of antihypertensive drugs on BP and its diurnal fluctuation. MetS hypertensive monkeys displayed higher BP, obesity, glucose intolerance, and dyslipidemia. We also confirmed impaired 24-h BP circadian rhythm in MetS hypertensive monkeys. Importantly, Eplerenone, a mineralocorticoid receptor blocker, exerts multiple beneficial effects in MetS hypertensive monkeys, including BP reduction, 24-h BP circadian rhythm restoration, and decreased plasma concentration of inflammation factors and advanced glycation end-products. In summary, we identified a naturally-developed hypertensive MetS NHP model, which is of great value in the studies on pathogenesis of MetS-associated hypertension and development of novel therapeutic strategies. We also provided multiple novel mechanistic insights of the beneficial effect of Eplerenone on MetS with hypertension.

Deficiency of PRKD2 triggers hyperinsulinemia and metabolic disorders

Hyperinsulinemia is the earliest symptom of insulin resistance (IR), but a causal relationship between the two remains to be established. Here we show that a protein kinase D2 (PRKD2) nonsense mutation (K410X) in two rhesus monkeys with extreme hyperinsulinemia along with IR and metabolic defects by using extreme phenotype sampling and deep sequencing analyses. This mutation reduces PRKD2 at both the mRNA and the protein levels. Taking advantage of a PRKD2-KO mouse model, we demonstrate that PRKD2 deletion triggers hyperinsulinemia which precedes to IR and metabolic disorders in the PRKD2 ablation mice. PRKD2 deficiency promotes β-cell insulin secretion by increasing the expression and activity of L-type Ca2+ channels and subsequently augmenting high glucose- and membrane depolarization-induced Ca2+ influx. Altogether, these results indicate that down-regulation of PRKD2 is involved in the pathogenesis of hyperinsulinemia which, in turn, results in IR and metabolic disorders.Hyperinsulinemia can precede the development of insulin resistance. Here the authors identify a PKD2 mutation that leads to hyperinsulinemia and insulin resistance in Rhesus monkey and show that PKD2 deficiency promotes beta cell insulin secretion by activating L-type Ca2+ channels.

Central role of RIPK1-VDAC1 pathway on cardiac impairment in a non-human primate model of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disorder characterized by destructive polyarthritis and systemic complications. It increases cardiovascular morbidity and mortality. However, the mechanism underlying RA-related cardiac damage remains largely unknown. Here, we found and characterized a non-human primate (NHP) model with spontaneous RA similar to the human conditions. Compared with the control group, the cardiac function in RA monkeys showed progressively deterioration; histologically, we found significantly increased inflammatory cell infiltration, cell death, and fibrosis in RA monkey heart tissue. Mechanistically, the upregulated receptor-interacting protein kinase 1 (RIPK1) in RA monkey heart tissue bound to voltage-dependent anion-selective channel 1 (VDAC1), increased VDAC1 oligomerization, and subsequently induced cardiac cell death and functional impairment. These findings identified that RIPK1-VDAC1 pathway is a promising target to treat cardiac impairment in RA. This unique model of RA will provide a valuable tool for mechanistic and translational studies.

Characterization of spontaneously-developed non-alcoholic fatty liver disease in aged rhesus monkeys

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a global epidemic afflicting 20–30% in the general population. The animal model of NAFLD available at the present are less clinically relevant. In this study. We aimed to establish a NAFLD model of rhesus monkeys and develop an ultrasonographic steatosis score (USS) system to grade hepatic steatosis in this model.MethodsWe performed hepatic ultrasonography and blood biochemical tests on 86 rhesus monkeys with and without metabolic syndrome (MetS), among which 45 animals were further assessed by histopathological analysis.ResultsThe liver histological features of rhesus monkeys NAFLD were resemble to those of NAFLD patients. There was a close correlation between the histological steatosis grade and the USS (Spearman’s coefficient, 0.705, p < 0.001). The USS sensitivity was 87.5% and the specificity was 94.6% when the cut-off was USS2. In addition, the prevalence of MetS was significantly higher in the USS2–3 group. Multiple risk factors of cardiometabolic disease, including obesity, insulin resistance and dyslipidemia were significantly correlated with the USS.ConclusionsNAFLD was developed spontaneously among aging in rhesus monkeys (with increased prevalence in the MetS monkeys), which provided an ideal model for NAFLD. The newly developed USS system can be used to evaluate fatty liver in the rhesus monkey. The model as well as the noninvasive assessment methodology will provide a powerful tool for mechanistic studies and preclinical test of novel therapies for NAFLD.