The impact of chronic mild hypoxia on cerebrovascular remodelling; uncoupling of angiogenesis and vascular breakdown

Background Chronic mild hypoxia (CMH, 8% O2) stimulates robust vascular remodelling in the brain, but it also triggers transient vascular disruption. This raises the fundamental question: is the vascular leak an unwanted side-effect of angiogenic remodelling or is it a pathological response, unrelated to endothelial proliferation, in which declining oxygen levels trigger endothelial dysfunction? Methods To answer this question, mice were exposed to CMH (8% O2) for periods up to 14 days, after which, brain tissue was examined by immunofluorescence (IF) to determine which type of blood vessel (arteriole, capillary or venule) was most commonly associated with endothelial proliferation and vascular leak and how this correlated with tight junction protein expression. Vascular perfusion was examined using DiI. Data were analysed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post-hoc test. Results The following was observed: (1) most endothelial proliferation and extravascular fibrinogen leak occurred in capillaries and to a lesser degree in venules, (2) much to our surprise, endothelial proliferation and extravascular fibrinogen leak never colocalized, (3) interestingly however, endothelial proliferation was strongly associated with an intravascular fibrinogen staining pattern not seen in stable blood vessels, (4) DiI perfusion studies revealed that angiogenic vessels were adequately perfused, suggesting that fibrinogen retention in angiogenic vessels is not due to temporary closure of the vessel, but more likely because fibrinogen is retained within the vessel wall, (5) bromodeoxyuridine (BrdU) labelling as a means to more permanently label proliferating endothelial cells, confirmed lack of any connection between endothelial proliferation and extravascular fibrinogen leak, while (6) in contrast, proliferating microglia were detected within extravascular leaks. Conclusions Taken together, our findings support the concept that in the short-term, hypoxia-induced endothelial proliferation triggers transient fibrinogen deposition within the walls of angiogenic blood vessels, but no overt vascular leak occurs in these vessels. Importantly, endothelial proliferation and extravascular fibrinogen leaks never co-localize, demonstrating that extravascular leak is not an unwanted side-effect of angiogenic endothelial proliferation, but rather a dysfunctional vascular response to hypoxia that occurs in a distinct group of non-angiogenic blood vessels.

A High Fidelity Transmural Anisotropic Ventricular Tissue Model Function to Investigate the Interaction Mechanisms of Drug: An In-Silico Model for Pharmacotherapy

A high fidelity transmural anisotropic ventricular tissue model consisting of endocardial, mid myocardial, and epicardial myocytes were configured to investigate drug interaction, such as Hydroxychloroquine (HCQ), under hypoxia conditions without and with pro-arrhythmic comorbidity like hypokalemia in (a) ventricular tissue b) its arrhythmogenesis for different dosages and (b) two different pacing sequences (Normal and tachycardiac). In-silico ventricular modeling indicates HCQ has an insignificant effect on hypoxia with and without comorbidities, except in the combination of mild hypoxia with moderate hypokalemia condition and severe hypoxia with mild hypokalemia where it initiated a re-entrant arrhythmia. Secondly, incorporating drug dosage variations indicates the 10 μM HCQ created PVCs for all settings except in severe hypoxia conditions where re-entrant arrhythmia occurred. In addition to the dosage of HCQ utilized for treatment, the pacing protocol also influences the appearance of re-entrant arrhythmia only for severe hypoxia with 10 μM HCQ dosage alone. For all other conditions, including tachycardiac pacing protocol, no arrhythmia occurred. These findings infer that the arrhythmic fatality rate due to HCQ treatment for hypoxia can be effectively alleviated by subtly altering or personalizing the dosage of HCQ and aid in the treatment of hypoxia-induced symptoms caused by COVID.

Influencing Factors of Daytime Sleepiness in Patients with Obstructive Sleep Apnea Hypopnea Syndrome and Its Correlation with Pulse Oxygen Decline Rate

Objective. To explore the influencing factors of daytime sleepiness in patients with obstructive sleep apnea hypopnea syndrome (OSAHS) and the correlation between daytime sleepiness and pulse oxygen decline rate in patients with severe OSAHS. Methods. From January 2018 to April 2021, 246 consecutive patients with OSAHS diagnosed by polysomnography (PSG) in our hospital were selected. All patients were grouped according to the minimum nocturnal oxygen saturation and apnea hypopnea index (AHI). There were 33 cases in the no sleep hypoxia group, 34 cases in the mild hypoxia group, 119 cases in the moderate hypoxia group, and 60 cases in the severe hypoxia group. There were 30 cases in the simple snoring group, 55 cases in the mild OSAHS group, 48 cases in the moderate OSAHS group, and 113 cases in the severe OSAHS group. The Epworth Sleepiness Scale (ESS) scores of each group were compared. All patients were grouped according to ESS score. Those with score ≥9 were included in the lethargy group (n = 118), and those with score ≤10 were included in the no lethargy group (n = 128). Univariate and multivariate logistic regression analyses were used to explore the influencing factors of daytime sleepiness in OSAHS patients. Pearson correlation analysis showed the correlation between ESS score and pulse oxygen decline rate in patients with severe OSAHS. Results. The ESS score of the severe hypoxia group > the moderate hypoxia group > the mild hypoxia group > the no sleep hypoxia group. There was significant difference among the groups (F = 19.700, P < 0.0001 ). There were significant differences between the severe hypoxia group and other groups and between the moderate hypoxia group and the no sleep hypoxia group and the mild hypoxia group ( P < 0.05 ). The ESS score of the severe OSAHS group > the moderate OSAHS group > the mild OSAHS group > the simple snoring group. There was significant difference among the groups (F = 19.000, P < 0.0001 ). There were significant differences between the severe OSAHS group and other groups and between the moderate OSAHS group and the simple snoring group ( P < 0.05 ). Univariate analysis showed that BMI, neck circumference, snoring degree, total apnea hypopnea time, AHI, micro arousal index (MAI), oxygen saturation (CT90%), lowest oxygen saturation (LSaO2), and mean oxygen saturation (MSaO2) were the influencing factors of daytime sleepiness in OSAHS patients ( P < 0.05 ). Multiple logistic regression analysis showed that AHI and CT90% were independent risk factors for daytime sleepiness in OSAHS patients ( P < 0.05 ). Pearson correlation analysis showed that there was a positive correlation between ESS score and pulse oxygen decline rate in patients with severe OSAHS (r = 0.765, P < 0.0001 ). Conclusion. OSAHS patients may be accompanied by daytime sleepiness in varying degrees, which may be independently related to AHI and CT90%. The degree of daytime sleepiness in patients with severe OSAHS may be closely related to the decline rate of pulse oxygen, which should be paid great attention in clinic.

BITC induces cardiomyocyte proliferation and heart regeneration

Mammalian cardiomyocytes have the ability to proliferate from the embryonic stage until early neonatal stage, with most of them being arrested from the cell cycle shortly after birth. Therefore, adult mammalian heart cannot regenerate myocardial injury. Despite much attention, pharmacological approaches for the induction of cardiomyocyte proliferation and heart regeneration have yet to be successful. To induce cardiomyocyte proliferation by drug administration, we focused on benzyl isothiocyanate (BITC). Firstly, we showed that BITC induces cardiomyocyte proliferation both in vitro and in vivo through the activation of the cyclin-dependent kinase (CDK) pathway. In addition, we demonstrated that BITC treatment induces heart regeneration in the infarcted neonatal heart even after the regeneration period. Furthermore, we administered BITC to adult mice in parallel with mild hypoxia (10% O2) treatment and showed that a combination of BITC administration and mild hypoxia exposure induces cell cycle reentry in the adult heart. The present study suggests that pharmacological activation of the CDK pathway with BITC concurrently with the activation of hypoxia-related signaling pathways may enable researchers to establish a novel strategy to induce cardiac regeneration in patients with heart disease.

The HIF1α-PDGFD-PDGFRα axis controls glioblastoma growth at normoxia/mild-hypoxia and confers sensitivity to targeted therapy by echinomycin

Background Glioblastoma multiforme (GBM), a lethal brain tumor, remains the most daunting challenge in cancer therapy. Overexpression and constitutive activation of PDGFs and PDGFRα are observed in most GBM; however, available inhibitors targeting isolated signaling pathways are minimally effective. Therefore, better understanding of crucial mechanisms underlying GBM is needed for developing more effective targeted therapies. Methods Target genes controlled by HIF1α in GBM were identified by analysis of TCGA database and by RNA-sequencing of GBM cells with HIF1α knockout by sgRNA-Cas9 method. Functional roles of HIF1α, PDGFs and PDGFRs were elucidated by loss- or gain-of-function assays or chemical inhibitors, and compared in response to oxygen tension. Pharmacological efficacy and gene expression in mice with intracranial xenografts of primary GBM were analyzed by bioluminescence imaging and immunofluorescence. Results HIF1α binds the PDGFD proximal promoter and PDGFRA intron enhancers in GBM cells under normoxia or mild-hypoxia to induce their expression and maintain constitutive activation of AKT signaling, which in turn increases HIF1α protein level and activity. Paradoxically, severe hypoxia abrogates PDGFRα expression despite enhancing HIF1α accumulation and corresponding PDGF-D expression. Knockout of HIF1A, PDGFD or PDGFRA in U251 cells inhibits cell growth and invasion in vitro and eradicates tumor growth in vivo. HIF1A knockdown in primary GBM extends survival of xenograft mice, whereas PDGFD overexpression in GL261 shortens survival. HIF1α inhibitor Echinomycin induces GBM cell apoptosis and effectively inhibits growth of GBM in vivo by simultaneously targeting HIF1α-PDGFD/PDGFRα-AKT feedforward pathway. Conclusions HIF1α orchestrates expression of PDGF-D and PDGFRα for constitutive activation of AKT pathway and is crucial for GBM malignancy. Therefore, therapies targeting HIF1α should provide an effective treatment for GBM.

Effects of Metformin on Spontaneous Ca2+ Signals in Cultured Microglia Cells under Normoxic and Hypoxic Conditions

Microglial functioning depends on Ca2+ signaling. By using Ca2+ sensitive fluorescence dye, we studied how inhibition of mitochondrial respiration changed spontaneous Ca2+ signals in soma of microglial cells from 5–7-day-old rats grown under normoxic and mild-hypoxic conditions. In microglia under normoxic conditions, metformin or rotenone elevated the rate and the amplitude of Ca2+ signals 10–15 min after drug application. Addition of cyclosporin A, a blocker of mitochondrial permeability transition pore (mPTP), antioxidant trolox, or inositol 1,4,5-trisphosphate receptor (IP3R) blocker caffeine in the presence of rotenone reduced the elevated rate and the amplitude of the signals implying sensitivity to reactive oxygen species (ROS), and involvement of mitochondrial mPTP together with IP3R. Microglial cells exposed to mild hypoxic conditions for 24 h showed elevated rate and increased amplitude of Ca2+ signals. Application of metformin or rotenone but not phenformin before mild hypoxia reduced this elevated rate. Thus, metformin and rotenone had the opposing fast action in normoxia after 10–15 min and the slow action during 24 h mild-hypoxia implying activation of different signaling pathways. The slow action of metformin through inhibition of complex I could stabilize Ca2+ homeostasis after mild hypoxia and could be important for reduction of ischemia-induced microglial activation.

Augmented Respiratory–Sympathetic Coupling and Hemodynamic Response to Acute Mild Hypoxia in Female Rodents With Chronic Kidney Disease

Carotid body feedback and hypoxia may serve to enhance respiratory–sympathetic nerve coupling (respSNA) and act as a driver of increased blood pressure. Using the Lewis polycystic kidney (LPK) rat model of chronic kidney disease, we examined respSNA in adult female rodents with CKD and their response to acute hypoxia or hypercapnia compared to Lewis control animals. Under urethane anesthesia, phrenic nerve activity, splanchnic sympathetic nerve activity (sSNA), and renal sympathetic nerve activity (rSNA) were recorded under baseline conditions and during mild hypoxic or hypercapnic challenges. At baseline, tonic SNA and blood pressure were greater in female LPK rats versus Lewis rats (all P &lt; 0.05) and respSNA was at least two-fold larger [area under the curve (AUC), sSNA: 7.8 ± 1.1 vs. 3.4 ± 0.7 μV s, rSNA: 11.5 ± 3 vs. 4.8 ± 0.7 μV s, LPK vs. Lewis, both P &lt; 0.05]. Mild hypoxia produced a larger pressure response in LPK [Δ mean arterial pressure (MAP) 30 ± 6 vs. 12 ± 6 mmHg] and augmented respSNA (ΔAUC, sSNA: 8.9 ± 3.4 vs. 2 ± 0.7 μV s, rSNA: 6.1 ± 1.2 vs. 3.1 ± 0.7 μV s, LPK vs. Lewis, all P ≤ 0.05). In contrast, central chemoreceptor stimulation produced comparable changes in blood pressure and respSNA (ΔMAP 13 ± 3 vs. 9 ± 5 mmHg; respSNA ΔAUC, sSNA: 2.5 ± 1 vs. 1.3 ± 0.7 μV s, rSNA: 4.2 ± 0.9 vs. 3.5 ± 1.4 μV s, LPK vs. Lewis, all P &gt; 0.05). These results demonstrate that female rats with CKD exhibit heightened respSNA coupling at baseline that is further augmented by mild hypoxia, and not by hypercapnia. This mechanism may be a contributing driver of hypertension in this animal model of CKD.

Effects of Normoxia, Hyperoxia, and Mild Hypoxia on Macro-Hemodynamics and the Skeletal Muscle Microcirculation in Anesthetised Rats

Objectives: Excessive oxygen (O2) administration may have a negative impact on tissue perfusion by inducing vasoconstriction and oxidative stress. We aimed to evaluate the effects of different inhaled oxygen fractions (FiO2) on macro-hemodynamics and microvascular perfusion in a rat model.Methods: Isoflurane-anesthetised spontaneously breathing male Wistar rats were equipped with arterial (carotid artery) and venous (jugular vein) catheters and tracheotomy, and randomized into three groups: normoxia (FiO2 21%, n = 6), hyperoxia (FiO2 100%, n = 6) and mild hypoxia (FiO2 15%, n = 6). Euvolemia was maintained by infusing Lactate Ringer solution at 10 ml/kg/h. At hourly intervals for 4 h we collected measurements of: mean arterial pressure (MAP); stroke volume index (SVI), heart rate (HR), respiratory rate (by means of echocardiography); arterial and venous blood gases; microvascular density, and flow quality (by means of sidestream dark field videomicroscopy on the hindlimb skeletal muscle).Results: MAP and systemic vascular resistance index increased with hyperoxia and decreased with mild hypoxia (p &lt; 0.001 in both cases, two-way analysis of variance). Hyperoxia induced a reduction in SVI, while this was increased in mild hypoxia (p = 0.002). The HR increased under hyperoxia (p &lt; 0.05 vs. normoxia at 3 h). Cardiax index, as well as systemic O2 delivery, did not significantly vary in the three groups (p = 0.546 and p = 0.691, respectively). At 4 h, microvascular vessel surface (i.e., the percentage of tissue surface occupied by vessels) decreased by 29 ± 4% in the hyperoxia group and increased by 19 ± 7 % in mild hypoxia group (p &lt; 0.001). Total vessel density and perfused vessel density showed similar tendencies (p = 0.003 and p = 0.005, respectively). Parameters of flow quality (microvascular flow index, percentage of perfused vessels, and flow heterogeneity index) remained stable and similar in the three groups.Conclusions: Hyperoxia induces vasoconstriction and reduction in skeletal muscle microvascular density, while mild hypoxia has an opposite effect.

Targeting the Mild-Hypoxia Driving Force for Metabolic and Muscle Transcriptional Reprogramming of Gilthead Sea Bream (Sparus aurata) Juveniles

On-growing juveniles of gilthead sea bream were acclimated for 45 days to mild-hypoxia (M-HYP, 40–60% O2 saturation), whereas normoxic fish (85–90% O2 saturation) constituted two different groups, depending on if they were fed to visual satiety (control fish) or pair-fed to M-HYP fish. Following the hypoxia conditioning period, all fish were maintained in normoxia and continued to be fed until visual satiation for 3 weeks. The time course of hypoxia-induced changes was assessed by changes in blood metabolic landmarks and muscle transcriptomics before and after exhaustive exercise in a swim tunnel respirometer. In M-HYP fish, our results highlighted a higher contribution of aerobic metabolism to whole energy supply, shifting towards a higher anaerobic fitness following normoxia restoration. Despite these changes in substrate preference, M-HYP fish shared a persistent improvement in swimming performance with a higher critical speed at exercise exhaustion. The machinery of muscle contraction and protein synthesis and breakdown was also largely altered by mild-hypoxia conditioning, contributing this metabolic re-adjustment to the positive regulation of locomotion and to the catch-up growth response during the normoxia recovery period. Altogether, these results reinforce the presence of large phenotypic plasticity in gilthead sea bream, and highlights mild-hypoxia as a promising prophylactic measure to prepare these fish for predictable stressful events.