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Sustained hyperglycemia results in excess protein O-GlcNAcylation, leading to vascular complications in diabetes. This study aims to investigate the role of O-GlcNAcylation in the progression of coronary microvascular disease (CMD) in inducible type 2 diabetic (T2D) mice generated by a high-fat diet with a single injection of low-dose streptozotocin. Inducible T2D mice exhibited an increase in protein O-GlcNAcylation in cardiac endothelial cells (CECs) and decreases in coronary flow velocity reserve (CFVR, an indicator of coronary microvascular function) and capillary density accompanied by increased endothelial apoptosis in the heart. Endothelial-specific O-GlcNAcase (OGA) overexpression significantly lowered protein O-GlcNAcylation in CECs, increased CFVR and capillary density, and decreased endothelial apoptosis in T2D mice. OGA overexpression also improved cardiac contractility in T2D mice. OGA gene transduction augmented angiogenic capacity in high-glucose treated CECs. PCR array analysis revealed that seven out of 92 genes show significant differences among control, T2D, and T2D + OGA mice, and Sp1 might be a great target for future study, the level of which was significantly increased by OGA in T2D mice. Our data suggest that reducing protein O-GlcNAcylation in CECs has a beneficial effect on coronary microvascular function, and OGA is a promising therapeutic target for CMD in diabetic patients.

Patients with diabetes with coronary microvascular disease (CMD) exhibit higher cardiac mortality than patients without CMD. However, the molecular mechanism by which diabetes promotes CMD is poorly understood. RNA-binding protein human antigen R (HuR) is a key regulator of mRNA stability and translation; therefore, we investigated the role of HuR in the development of CMD in mice with type 2 diabetes. Diabetic mice exhibited decreases in coronary flow velocity reserve (CFVR; a determinant of coronary microvascular function) and capillary density in the left ventricle. HuR levels in cardiac endothelial cells (CECs) were significantly lower in diabetic mice and patients with diabetes than the controls. Endothelial-specific HuR-KO mice also displayed significant reductions in CFVR and capillary density. By examining mRNA levels of 92 genes associated with endothelial function, we found that HuR, Cx40, and Nox4 levels were decreased in CECs from diabetic and HuR-KO mice compared with control mice. Cx40 expression and HuR binding to Cx40 mRNA were downregulated in CECs from diabetic mice. Cx40-KO mice exhibited decreased CFVR and capillary density, whereas endothelium-specific Cx40 overexpression increased capillary density and improved CFVR in diabetic mice. These data suggest that decreased HuR contributes to the development of CMD in diabetes through downregulation of gap junction protein Cx40 in CECs.

Background Abnormal endothelial function in the lungs is implicated in the development of pulmonary hypertension; however, there is little information about the difference of endothelial function between small distal pulmonary artery (PA) and large proximal PA and their contribution to the development of pulmonary hypertension. Herein, we investigate endothelium-dependent relaxation in different orders of PAs and examine the molecular mechanisms by which chronic hypoxia attenuates endothelium-dependent pulmonary vasodilation, leading to pulmonary hypertension. Methods and Results Endothelium-dependent relaxation in large proximal PAs (second order) was primarily caused by releasing NO from the endothelium, whereas endothelium-dependent hyperpolarization (EDH)-mediated vasodilation was prominent in small distal PAs (fourth-fifth order). Chronic hypoxia abolished EDH-mediated relaxation in small distal PAs without affecting smooth muscle-dependent relaxation. RNA-sequencing data revealed that, among genes related to EDH, the levels of Cx37, Cx40, Cx43, and IK were altered in mouse pulmonary endothelial cells isolated from chronically hypoxic mice in comparison to mouse pulmonary endothelial cells from normoxic control mice. The protein levels were significantly lower for connexin 40 (Cx40) and higher for connexin 37 in mouse pulmonary endothelial cells from hypoxic mice than normoxic mice. Cx40 knockout mice exhibited significant attenuation of EDH-mediated relaxation and marked increase in right ventricular systolic pressure. Interestingly, chronic hypoxia led to a further increase in right ventricular systolic pressure in Cx40 knockout mice without altering EDH-mediated relaxation. Furthermore, overexpression of Cx40 significantly decreased right ventricular systolic pressure in chronically hypoxic mice. Conclusions These data suggest that chronic hypoxia-induced downregulation of endothelial Cx40 results in impaired EDH-mediated relaxation in small distal PAs and contributes to the development of pulmonary hypertension.

AimsWe previously reported that increased protein O-GlcNAcylation in diabetic mice led to vascular rarefaction in the heart. In this study, we aimed to investigate whether and how coronary endothelial cell (EC) apoptosis is enhanced by protein O-GlcNAcylation and thus induces coronary microvascular disease (CMD) and subsequent cardiac dysfunction in diabetes. We hypothesize that excessive protein O-GlcNAcylation increases p53 that leads to CMD and reduced cardiac contractility.Methods and resultsWe conducted in vivo functional experiments in control mice, TALLYHO/Jng (TH) mice, a polygenic type 2 diabetic (T2D) model, and EC-specific O-GlcNAcase (OGA, an enzyme that catalyzes the removal of O-GlcNAc from proteins)-overexpressing TH mice, as well as in vitro experiments in isolated ECs from these mice. TH mice exhibited a significant increase in coronary EC apoptosis and reduction of coronary flow velocity reserve (CFVR), an assessment of coronary microvascular function, in comparison to wild-type mice. The decreased CFVR, due at least partially to EC apoptosis, was associated with decreased cardiac contractility in TH mice. Western blot experiments showed that p53 protein level was significantly higher in coronary ECs from TH mice and T2D patients than in control ECs. High glucose treatment also increased p53 protein level in control ECs. Furthermore, overexpression of OGA decreased protein O-GlcNAcylation and down-regulated p53 in coronary ECs, and conferred a protective effect on cardiac function in TH mice. Inhibition of p53 with pifithrin-α attenuated coronary EC apoptosis and restored CFVR and cardiac contractility in TH mice.ConclusionsThe data from this study indicate that inhibition of p53 or down-regulation of p53 by OGA overexpression attenuates coronary EC apoptosis and improves CFVR and cardiac function in diabetes. Lowering coronary endothelial p53 levels via OGA overexpression could be a potential therapeutic approach for CMD in diabetes.

Background and purposeChloroquine is a traditional medicine to treat malaria. There is increasing evidence that chloroquine not only induces phagocytosis but regulates vascular tone. Few reports investigating the effect of chloroquine on vascular responsiveness of coronary arteries have been made. In this study, we examined how chloroquine affected endothelium-dependent relaxation in coronary arteries under normal and diabetic conditions.Experimental approachWe isolated coronary arteries from mice and examined endothelium-dependent relaxation (EDR). Human coronary endothelial cells and mouse coronary endothelial cells isolated from control and diabetic mouse (TALLYHO/Jng [TH] mice, a spontaneous type 2 diabetic mouse model) were used for the molecular biological or cytosolic NO and Ca2+ measurements.Key resultsChloroquine inhibited endothelium-derived NO-dependent relaxation but had negligible effect on endothelium-derived hyperpolarization (EDH)-dependent relaxation in coronary arteries of control mice. Chloroquine significantly decreased NO production in control human coronary endothelial cells partly by phosphorylating eNOSThr495 (an inhibitory phosphorylation site of eNOS) and attenuating the rise of cytosolic Ca2+ concentration after stimulation. EDR was significantly inhibited in diabetic mice in comparison to control mice. Interestingly, chloroquine enhanced EDR in diabetic coronary arteries by, specifically, increasing EDH-dependent relaxation due partly to its augmenting effect on gap junction activity in diabetic mouse coronary endothelial cells.Conclusions and implicationsThese data indicate that chloroquine affects vascular relaxation differently under normal and diabetic conditions. Therefore, the patients' health condition such as coronary macrovascular or microvascular disease, with or without diabetes, must be taken account into the consideration when selecting chloroquine for the treatment of malaria.

The article presents a case study of an 11-year-old boy with autosomal dominant hypocalcemia (ADH) treated with vitamin D and thiazide, who experienced a severe hypercalcemic episode. Topics discussed include the patient's treatment history, the development of hypercalcemia, and the potential association with a milk-alkali syndrome-like syndrome triggered by increased dietary calcium intake.

The sex-determining region of the Y chromosome, Sry/SRY, is an initiation factor for testis development in both humans and mice. Although the functional compatibility between murine SRY and human SRY was previously examined in transgenic mice, their equivalency remains inconclusive. Because molecular interaction and timeline of mammalian sex determination were mostly described in murine experiments, we generated a mouse model in which Sry was substituted with human SRY to verify the compatibility. The mouse model had the human SRY open reading frame at the locus of murine Sry exon 1— Sry (SRY) mice—and was generated using the CRISPR/Cas9 system. The reproductive system of the mice was analyzed. The expression of human SRY in the fetal gonadal ridge of Sry (SRY) mice was detected. The external and internal genitalia of adult Sry (SRY) mice were similar to those of wild-type females, without any significant difference in anogenital distance. Sry (SRY) mice obtained gonads, which were morphologically considered as ovaries. Histological analysis revealed that the cortical regions of gonads from adult Sry (SRY) mice contained few follicles. We successfully replaced genes on the Y chromosome with targeted genome editing using the CRISPR/Cas9 system. Because the Sry (SRY) XY mice did not develop testis, we concluded that human SRY was insufficient to drive testis development in mouse embryos. The difference in response elements and lack of glutamine-rich domains may have invalidated human SRY function in mice. Signal transduction between Sry / SRY expression and Sox9 / SOX9 activation is possibly organized in a species-specific manner. [ABSTRACT FROM AUTHOR]

Background: One of the major purposes of newborn screening for 21‐hydroxylase deficiency (21OHD) is preventing life‐threatening adrenal crisis. However, the details of adrenal crisis in newborns are not precisely documented. Aim: We aimed to clarify the clinical details of salt‐wasting in newborn 21OHD patients. Methods: Based on the follow‐up survey of the screening in Tokyo from 1989 to 2017, we retrospectively analysed the conditions of classical 21OHD neonates before the initiation of therapy. Results: One hundred classical 21OHD patients (55 male, 45 female) were analysed. The age at the first hospital visit was 0–20 days with sex difference (male: 9.0 ± 3.5 days; female: 6.2 ± 3.9 days). Thirty‐seven (37.4%) patients exhibited severe salt‐wasting (SSW), that is, Na < 130 mEq/L, K > 7 mEq/L or Na/K ratio < 20; except for one case, SSW developed in or after the second week of life. The serum concentrations of Na, K and Na/K were linearly correlated with age in days (R2 =.38,.25, and.34 respectively), suggesting that the risk of SSW increases linearly without a threshold. The age at which the regression lines reached Na < 130 mEq/L, K > 7 mEq/L and Na/K < 20 was approximately coincided, 11.1, 12.3 and 11.2 days, respectively. All SSW patients exhibited decreased body weight from birth in their second week of life. Conclusion: Our data revealed that the risk of developing SSW increases during the second week of life without a threshold, and for preventing SSW, early intervention, ideally during first week of life, is desirable. An increased body weight in the second week of life indicates the absence of SSW. [ABSTRACT FROM AUTHOR]

Metabolic syndrome is a critically important precursor to the onset of many diseases, such as cardiovascular disease, and cardiovascular disease is the leading cause of death worldwide. The primary risk factors of metabolic syndrome include hyperglycaemia, abdominal obesity, dyslipidaemia, and high blood pressure. It has been well documented that metabolic syndrome alters vascular endothelial and smooth muscle cell functions in the heart, brain, kidney and peripheral vessels. However, there is less information available regarding how metabolic syndrome can affect pulmonary vascular function and ultimately increase an individual's risk of developing various pulmonary vascular diseases, such as pulmonary hypertension. Here, we review in detail how metabolic syndrome affects pulmonary vascular function. Potential mechanisms underlining pulmonary vascular dysfunction caused by metabolic syndrome and subsequent increase in susceptibility to pulmonary hypertension. [ABSTRACT FROM AUTHOR]

Background: Type A insulin resistance (IR) is a rare form of severe congenital IR that is frequently caused by heterozygous mutations in the insulin receptor (INSR) gene. Although Type A IR requires appropriate intervention from the early stages of diabetes, proper diagnosis of this disease is challenging, and accumulation of cases with detailed clinical profiles and genotypes is required. Methods: Herein we report on six peripubertal patients with clinically diagnosed Type A IR, including four patients with an identified INSR mutation. To clarify the clinical features of Type A IR due to INSR mutation, we validated the clinical characteristics of Type A IR patients with identified INSR mutations by comparing them with mutation‐negative patients. Results: Four heterozygous missense mutations within the β‐subunit of INSR were detected: Gly1146Arg, Arg1158Trp, Arg1201Trp, and one novel Arg1201Pro mutation. There were no obvious differences in clinical phenotypes, except for normal lipid metabolism and autosomal dominant inheritance, between Type A IR due to INSR mutations and Type A IR due to other factors. However, our analysis revealed that the extent of growth retardation during the fetal period is correlated with the severity of insulin signaling impairment. Conclusions: The present study details the clinical features of four patients with genetically proven Type A IR. Further accumulation of genetically proven cases and long‐term treatment prognoses following early diagnosis are required to further elucidate the dynamics of this disease. HighlightsThis study presents details of six peripubertal cases with clinically diagnosed Type A insulin resistance (IR), including four cases with identified insulin receptor (INSR) gene mutations (three previously reported and one novel).The study reveals the clinical features of patients with Type A IR due to INSR mutations compared with mutation‐negative patients, and indicates that the extent of growth retardation during the fetal period is correlated with the severity of insulin signaling impairment. [ABSTRACT FROM AUTHOR]

Background: MECP2 duplication syndrome, which is caused by duplication of part of the Xq28 region containing the MECP2 gene, causes intellectual disability and mild dysmorphic features in males. To date, precocious puberty has not been reported as a clinical feature of MECP2 duplication syndrome. Methods: A 6-year-old male with severe intellectual disability was referred because of growth acceleration and precocious puberty. We checked his hormonal profile and conducted imaging studies and an array comparative genomic hybridization analysis. Results: His bone age (9 years and 6 months) was accelerated, and the basal level of testosterone was 8.99 ng/ml. In a luteinizing hormone (LH)-releasing hormone (LHRH) stimulation test, LH increased from 3.69 to 9.32 IU/l, and follicle-stimulating hormone increased from 0.65 to 0.90 IU/l. Chest and abdominal CTs and a brain MRI did not reveal any abnormalities. Treatment with an LHRH analogue effectively suppressed the level of testosterone to <0.03 ng/ml, consistent with the diagnosis of gonadotropin-dependent precocious puberty (GDPP). We identified a duplication of the Xq28 locus including MECP2 in the patient. Conclusion: Precocious puberty is often a benign central process in girls, but it is rarely idiopathic in boys. The present case raises the possibility that GDPP is a novel clinical feature of MECP2 duplication syndrome. [ABSTRACT FROM AUTHOR]

Epidemiological data reveal that diabetic patients exhibit higher incidence of pulmonary hypertension (PH) than control subjects. We also reported that PH patients with diabetes exhibit increased pulmonary arterial stiffness and elastance, and decreased pulmonary artery capacitance in comparison to PH patients without diabetes. However, little is known whether and how diabetes alters the susceptibility to pulmonary hypertension. In this study, we examine the effect of diabetes on the development of hypoxic-induced pulmonary hypertension (HPH). We used inducible T2D (iT2D) mice and genetically modified T2D mice (KKAy mice). iT2D mice were generated by a single injection of low dose streptozotocin (75mg/kg, i.p.) with a high fat diet (60 % kcal from fat). Chronic hypoxia was induced by placing mice in the hypoxic chamber (10% O2) for 4 weeks. iT2D mice in hypoxia (DH) showed more severe HPH than the control in hypoxia (CH), determined by a measurement of right ventricular systolic pressure (RVSP). Hypoxic exposure also increased RVSP in KKAy mice greater than in wild type mice. There was no difference of pulmonary arterial branch numbers and vascular contractility in pulmonary artery (PA) between CH and DH mice. However, endothelium-dependent relaxation (EDR) in PAs was significantly suppressed in DH mice compared to CH mice. Attenuated EDR in DH mice was restored by the pretreatment of mitoTEMPOL (a mitochondrial O2- scavenger) toward the level seen in CH mice, suggesting that increased mitochondrial reactive oxygen species ([ROS]mito) might be involved in attenuated EDR in DH mice. We then isolated mouse pulmonary endothelial cells (MPECs) from CH and DH mice and determine the level of [ROS]mito. MPECs from DH mice exhibited significant increase in [ROS]mito compared to MPECs of CH mice. Furthermore, we found that the protein level of mitochondrial superoxide dismutase (SOD2) in MPECs was significantly lower in DH mice than CH mice. Our results suggest that decreased SOD2 in MPECs of DH mice increases [ROS]mito, attenuates EDR in PAs, and resulted in more severe HPH in diabetes. Targeting mitochondrial ROS may be a potential therapy for HPH in diabetic population. [ABSTRACT FROM AUTHOR]

Coronary artery disease and coronary microvascular disease are a leading cause of death in diabetic patients. However, the molecular mechanisms by which diabetes causes coronary microvascular dysfunction is not well understood. Protein O -GlcNAcylation is one of the posttranslational modifications and regulated by two enzymes; O -GlcNAc transferase catalyzes the addition of O -GlcNAc to the proteins and O -GlcNAcase (OGA) removes O -GlcNAc. We recently reported that increased protein O -GlcNAcylation attenuated endothelium-dependent vascular relaxation in type 1 diabetic mice. In this study, we investigate whether and how excessive protein O -GlcNAcylation leads to coronary microvascular dysfunction in type 2 diabetic (T2D) mice. We used endothelium-specific tetracycline-inducible OGA transgenic mice. T2D mice were generated by a single injection of low dose streptozotocin (75mg/kg, i.p.) with a high fat diet (60 % kcal from fat). Six weeks after T2D induction, doxycycline was administered in drinking water (0.2 mg/ml) for 6 weeks. T2D mice exhibited decreased coronary flow velocity reserve (CFVR, a marker of coronary microvascular dysfunction), increased EC apoptosis and decreased capillary density in the left ventricle (LV) compared to the control. OGA overexpression in T2D mice restored the level of EC apoptosis, capillary density, and CFVR toward control levels. T2D mice also exhibited attenuated cardiac contractility evidenced by decreased left ventricular systolic pressure, ejection fraction, and dp/dt. These parameters were increased by OGA overexpression in T2D mice. To investigate the target genes influenced by protein O -GlcNAcylation, we isolated mouse coronary ECs and performed a PCR Array. We revealed that Sp1 and Mapk1 were decreased in T2D mice and increased by OGA overexpression in T2D. Our results suggest that increased protein O -GlcNAcylation leads to coronary microvascular disease via increasing EC apoptosis. Overexpression of OGA in ECs not only restores coronary microvascular function, but also improves cardiac function. Further study on the role of Sp1 and Mapk1 in coronary microvascular disease might help develop novel treatment for coronary microvascular disease and cardiac dysfunction in type 2 diabetic patients. [ABSTRACT FROM AUTHOR]

The nuclear receptor NR5A1 is equally expressed and required for development of the gonadal primordia of both sexes, but, after sex determination, it is upregulated in XY testes and downregulated in XX ovaries. We have recently demonstrated, in mice, that this downregulation is mediated by forkhead box L2 (FOXL2) and hypothesized that adequate suppression of Nr5a1 is essential for normal ovarian development. Further, analysis of human patients with disorders/differences of sex development suggests that overexpression of NR5A1 can result in XX (ovo)testicular development. Here, we tested the role of Nr5a1 by overexpression in fetal gonads using a Wt1 -BAC (bacterial artificial chromosome) transgene system. Enforced Nr5a1 expression compromised ovarian development in 46,XX mice, resulting in late-onset infertility, but did not induce (ovo)testis differentiation. The phenotype was similar to that of XX mice lacking Notch signaling. The expression level of Notch2 was significantly reduced in Nr5a1 transgenic mice, and the ovarian phenotype was almost completely rescued by in utero treatment with a NOTCH2 agonist. We conclude that suppression of Nr5a1 during the fetal period optimizes ovarian development by fine-tuning Notch signaling., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)