bluuebunny/crossref_metadata_2025 huggingface dataset:
Upregulation of Orexin Receptor in Paraventricular Nucleus Promotes Sympathetic Outflow Through Non‐selective Cation Channel in Obesity
Authors: Zhou Jing‐Jing , Chen Shao‐Rui , Yuan Fang , Zhang Yi , Li De‐Pei
Published: June 2021

Elevated sympathetic outflow contributes to hypertension in obesity. Orexins importantly regulates autonomic function. Presently we determined the role of orexin in the paraventricular (PVN) nucleus of hypothalamus in controlling sympathetic outflow in obese Zucker rats (OZRs) and lean Zucker rats (LZRs). Orexin A microinjection into PVN significiantly dose‐dependently elevated arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA) manner in anesthetized OZRs than LZRs, which eliminated by SB‐334867, orexin receptor 1 receptor (OX1R) antagonist, but not TCS‐OX2‐29, OX2R antagonist. Furthermore, SB‐334867 reduced basal ABP and RSNA in OZRs but not LZRs. Compared with LZRs, OX1R expression in PVN was significantly higher in OZRs, while no difference for OX2R. OX1R immunoreactivity was positive in retrogradely labeled PVN‐spinal neurons. Compared with LZRs, the basal firing rate in the PVN‐spinal neurons was higher in OZRs, and orexin A excited firing activity in PVN‐spinal neurons in OZRs, which eliminated by SB334867. SB334867 also decreased basal firing activity of these neurons in OZRs. In addition, orexin A induced larger currents in PVN spinal neurons in OZRs. These data suggest that upregulation of OX1R in the PVN promotes hyperactivity of PVN presympathetic neurons and sympathetic outflow in obesity.

This work was supported by National Institutes of Health (Grants MH096086 to D.‐P. L.) and National Natural Sciences Foundation of China (31271223 and 31071002).
Electrical Stimulation of Carotid Sinus in Conscious Normotensive and Spontaneously Hypertensive Rats
Authors: Domingos Gean , Santos‐Almeida Fernanda , Silva Luiz Eduardo , Dias Daniel , da Silva Carlos Alberto , Castania Jaci , Salgado Helio , Fazan Rubens
Published: June 2021

Electrical stimulation (ES) of the carotid sinus (CS) has been used to treat resistant arterial hypertension. Our aim was to evaluate, in conscious normotensive and spontaneously hypertensive rats (SHR), the effect of sustained (60 min) ES of CS on arterial pressure (AP), heart rate (HR) and heart rate variability (HRV). Male Wistar rats (n=8) and SHR (n=8) had the left CS surrounded by a pair of electrodes and insulated from adjacent tissues. A catheter was introduced into the femoral artery and exteriorized together with the CS electrodes. On the next day, conscious rats under continuous recording of AP had their CS stimulated with square pulses of 1 mA, 1 ms, and 30 Hz, delivered randomly, continuously or intermittently (20/20 s ON/OFF), during 60 min. On the following day the type of CS stimulation was inverted. To analyze HRV, segments of 5 min were selected immediately before, at the onset, middle and end of ES period. Greater sustained fall in AP due to ES of CS was observed in SHR (‐50±7 vs ‐11±2 mmHg). A conspicuous bradycardia was also elicited by sustained ES of the CS (‐62±15 and ‐32±11 bpm in SHR and Wistar rats). No difference was observed between continuous or intermittent ES of CS. Overall, HRV was increased by ES of CS only in SHR (from 4.8±0.6 to 11.7±3.2, 7.0±0.6 and 6.7±1.0 bpm at the onset, middle and end of ES). Surprisingly, no differences were observed in HR spectra during ES of CS in both groups. We conclude that ES of the CS was more effective to reduce AP and HR in SHR as compared to normotensive rats. Moreover, despite a marked effect of ES in overall HRV in SHR, no changes in HR spectra were observed.

Financial Support: FAPESP, CAPES and CNPq.
Chronic Heart Failure Blunts the Low Pressure Baroreflex Induced Biphasic Change of Sympathetic Nerve Activity
Authors: Oga Yasuhiro , Kishi Takuya , Saku Keita , Kakino Takamori , Nishizaki Akiko , Arimura Takahiro , Fujii Kana , Tohyama Takeshi , Nishikawa Takuya , Sunagawa Kenji
Published: June 2021

Background

We previously showed that volume loading activates low pressure baroreflex (L‐BARO) and causes biphasic change in sympathetic nerve activity (SNA). We examine if chronic heart failure (CHF) blunts the L‐BARO regulation of SNA.

Methods

We used 10 anesthetized Sprague‐Dawley rats. CHF was induced by myocardial infarction. We vascularly isolated carotid sinuses and kept carotid sinus pressure constant to abolish the arterial baroreflex which is the major regulator of SNA. We infused or withdrew blood (3ml/kg) stepwise to activate L‐BARO and recorded SNA and hemodynamics.

Results

In the control group (n=5), volume loading evoked biphasic changes in SNA with a maximal response of 61±43%. In contrast, CHF (n=5) markedly reduced the maximum response (10±13% from baseline) and blunts L‐BARO induced SNA alteration. In both group, vagotomy totally abolished the L‐BARO regulation of SNA. Regarding hemodynamics, L‐BARO attenuated the increases in AP and LV end‐diastolic pressure (LVEDP) against volume infusion in control group, whereas did not affect either in CHF.

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Conclusions

CHF blunted L‐BARO modulation of SNA in rats. The mechanism and pathophysiological significance of L‐BARO failure in CHF remains to be seen.
Developmental Exposure to Indoor Flame Retardants Disrupts Sympathetic and Hypothalamic‐Pituitary‐Adrenal (HPA) Axis Activity in Osmotic Challenged Rats
Authors: Gutierrez Roberto , Spurgin Kurt , Prien Alex , Curras‐Collazo Margarita
Published: June 2021

Research in our lab has demonstrated that perinatal exposure to the polybrominated‐dipheyl ether (PBDE) mixture, DE‐71, triggers pressor responses to hyperosmotic stress in adult rats. To evaluate the effect of ganglionic blockade (GB), dams were dosed with DE‐71 or corn‐oil daily from gestational day 4 through postnatal day (PND) 21. At PND 60 offspring received hyperosmotic (3.5 M NaCl) or normosmotic NaCl (0.9g%) i.p. with or without pentolinium (19.2mg/kg). After 3 hours, blood pressure was measured as % Δ baseline using sphygmomanometry under anesthesia. Hyperosmotic treatment produced a significant increase in systolic BP in PBDE‐rats compared to oil‐controls (22.99+ 2.55 vs 1.53+2.25%, n=22; p<0.001). GB partially occluded the pressor response in PBDE hyperosmotic rats, (12.85+1.30%, n=12; p<0.01), suggesting involvement of sympathetic nervous system (SNS). Adrenal catecholamine (CA) content, measured via fluorometric detection, was significantly reduced in PBDE hyperosmotic vs normosmotic rats (5.4+1.3 vs. 1.3+0.4 mM; n=21, p<0.05), suggesting exaggerated CA release. This was not compensated for by increased mRNA levels for TH, PNMT or PACAP mRNA. Plasma corticosterone was significantly elevated in PBDE hyperosmotic rats vs controls suggesting HPA overactivation. Adrenal HPA markers appeared to rise in the PBDE hyperosmotic rats, but this was not significant.In conclusion, adult hyperosmotic treatment in rats exposed to PBDEs perinatally show disrupted SNS and CA balance and hyperactive HPA axis. Our findings reveal that PBDEs may target endocrine and autonomic systems associated with stress responses.
Mean arterial pressure and wave reflection during the cold pressor test: the influence of trunk fat
Authors: Harwood Morton , Wakeham Travis , Durocher John
Published: June 2021

Previous work reports that changes in the aortic augmentation index (AIx) are correlated with changes in mean arterial pressure (MAP) during the cold pressor test (CPT) in normal weight adults. The purpose of the present study is to examine the relationship between MAP and AIx responses during CPT in those with high (蠅32%; n=10) and low (蠄 30%; n=10) trunk fat. Trunk fat was determined via bioelectric impedance analysis (Tanita BC‐418). Participants were tested in the supine position during a 3 min baseline and 3 min CPT while MAP and heart rate (HR) were continuously recorded. Radial applanation tonometry measurements were assessed approximately 30 s into each minute, and AIx is reported as a mean of baseline measurements and from the 3rd minute of CPT (n=7 for each group). Baseline MAP, HR, and AIx were similar in the 2 groups. The increases in MAP (Δ18±3 vs. Δ17±3 mmHg), HR (Δ9±1 vs. Δ4±2 beats/min), and AIx (Δ14±5 vs. Δ9±5%) were similar for the low and high trunk fat groups, respectively. Consistent with previous work the MAP responses to CPT were correlated to changes in AIx across all participants (r=0.70; p<0.01). When separately examining the low trunk fat group, ΔMAP and ΔAIx remained significantly correlated (r=0.87; p<0.01), but in the high fat group the correlation was no longer significant (r=0.43; p=0.33). Our results indicate that while MAP responses to CPT are similar in low and high trunk fat participants, the relationship between ΔMAP and ΔAIx appears to be disrupted by elevated trunk fat.
Reconstitution of WKY with SHR Bone Marrow Alters Vascular Hemodynamics and Sympathetic Drive
Authors: Santisteban Monica , Garcia‐Pereira Fernando , Ahmari Niousha , Marulanda Jessica , Johnson Richard , Raizada Mohan , Zubcevic Jasenka
Published: June 2021

Our studies have established that the bone marrow (BM) of spontaneously hypertensive rats (SHR) is pro‐inflammatory. Reconstitution of WKY rat with SHR BM results in a persistent increase in mean arterial pressure (MAP) and increase in both peripheral and neuroinflammation. In the present study we tested the hypothesis that these changes are associated with alterations in vascular hemodynamics and sympathetic tone. WKY rats underwent BM reconstitution with either WKY or SHR BM (WKY‐WKY n=6; WKY‐SHR n=5). Spectral analysis was performed from continuous telemetry recordings. Blood perfusion in hind paws was determined by PeriCam PSI system for 5 minutes under 2% isoflurane. The WKY‐SHR rats had elevated MAP (147±16 vs 114±2 mmHg; p<0.05) and increased plasma NE (3131±520 vs 937±671 pg/ml; p<0.05) compared to the WKY‐WKY. This was associated with elevated sympathetic tone [LF(SBP): 7.1±0.6 vs 5.0±0.4 ms2/mmHg2; VLF(SBP): 3.7±0.2 vs 3.0±0.2 ms2/mmHg2; p<0.05] and dampened spontaneous baroreflex gain in the WKY‐SHR [sBRG(PI) 0.58±0.05 vs 0.87±0.09 ms/mmHg; p<0.05]. In addition, the rate of increased perfusion over time was lower in the WKY‐SHR rats (45.3±6.2 vs 18.5±8.0 AU intensity/minutes; p<0.05). Stabilized perfusion did not change. These data indicate that, in addition to increasing MAP, reconstitution with the BM cells from the SHR worsens cardiovascular variables both centrally and peripherally. They support the concept that pro‐inflammatory BM cells play a critical role in increased sympathetic tone and decreased perfusion, both key events in the development of persistent HTN.
The Effect of Multiple Sclerosis on Carotid Baroreflex Control of Heart Rate and Blood Pressure
Authors: Huang Mu , Allen Dustin , Keller David , Davis Scott
Published: June 2021

Multiple sclerosis (MS), a progressive neurological disease, can result in impairments in autonomic control of cardiovascular function. We tested the hypothesis that patients with MS (n=5) exhibit a blunted response following baroreflex perturbation compared to sex and age matched healthy controls (CON: n=5). At rest, 5‐sec pulses of neck suction (NS; ‐60 Torr) and neck pressure (NP; +40 Torr) were applied to simulate carotid hypertension and hypotension, respectively. Mean arterial pressure (MAP; finger photoplethysmography) and heart rate (HR) were continuously measured in response to the perturbations. Following NS, responses in MAP (MS: ‐11±5 mmHg, CON: ‐10±3 mmHg; p=0.59) and HR (MS: ‐8±3 BPM, CON: ‐8±4 BPM; p=0.93) were similar between groups. While HR response (MS: 5±5 BPM, CON: 5±4 BPM; p=0.94) following NP was no different, a difference in MAP approached significance (MS: 6±2 mmHg, CON: 8±2 mmHg; p=0.09). These data suggest that patients with MS demonstrate a diminished ability to increase MAP to hypotensive stimuli compared to healthy controls.
Supported by NIH R15HL117224 and National MS Society RG4696A
Maternal Obesity and the Developmental Programming of Hypertension: The role of Leptin in the Central Nervous System
Authors: Lim Joon (Kyungjoon) , Burke Sandra , Head Geoffery
Published: June 2021

The obesity in women among child baring age is increasing and this has been reported to be parallel to the increase in obesity in general population. We determined the trans‐generational ‘programming’ of leptin signalling in the central nervous system (CNS) following an initiation of a high fat diet (HFD) in mothers.
Female New Zealand White rabbits were fed a high fat (13%) diet (mHFD) or a normal fat diet (mNFD) prior mating and during pregnancy. Offspring from mNFD rabbits were subdivided and fed a HFD for 10days (mNFD10dHFD) to mimic adulthood obesity. All rabbits received an intracerebroventricular (ICV) catheter into the lateral ventricle and a recording electrode on the left renal nerve. Experiments were conducted in conscious rabbits and animals received increasing doses of melanocortin receptor antagonist (SHU9119), α‐Melanocortin stimulating hormone (αMSH) and a single dose of Leptin antagonist via ICV. BP, HR and RSNA were measured.
ICV SHU9119 reduced BP (‐5.8±0.7mmHg and ‐4.1±0.9mmHg) and RSNA (‐2.4±0.3 nu and ‐0.7±0.3 nu) in mHFD and mNFD10dHFD rabbits (P<0.001). Leptin antagonist reduced BP and RSNA only in mHFD rabbits (‐2.1±0.5mmHg and ‐2.7nu, respectively). αMSH injection increased BP, HR and RSNA in both mHFD and mNFD10dHFD rabbits (P<0.05). Total % fat was increased (50%) in all rabbits that had HFD.
Obesity during pregnancy ‘programs' leptin signalling pathway in the CNS of the offspring during development. Leptin via activation of melanocirtin pathway plays a key role in the CNS contributing to the pressor and tachycardic effects as well as renal sympathetic nerve activity in the pathophysiology of obesity.
Adaptive Plasticity in the Spinal Control of Left‐right Coordination During Locomotion
Authors: Frigon Alain , Dambreville Charline , Thibaudier Yann , Hurteau Marie‐France , Kuczynski Victoria , Rhodes Emily
Published: June 2021

Hindlimb (HL) locomotion recovers in chronic spinal‐transected cats following ankle extensors neurectomy (Bouyer et al. 2001), indicating compensatory spinal mechanisms. However, such recovery has only been tested during single‐belt treadmill locomotion at moderate speeds (0.35 m/s to 0.5 m/s). Here, HL locomotion was assessed at several speeds and left‐right speed differences during tied‐belt (equal left‐right speeds) and split‐belt (unequal left‐right speeds) locomotion, respectively, in 4 chronic spinal‐transected cats implanted with chronic electrodes for EMG. Spinal transection was made at the last thoracic segments and cats were trained to recover HL locomotion. A unilateral denervation of the lateral gastrocnemius and soleus was made after stable HL locomotion had recovered, at least 1 month post‐transection. Denervation produced variable results between cats. In the most affected cat, the denervated HL made abnormal contacts with the paw dorsum and left and right HLs were not coordinated 1‐2 days post‐denervation, producing an unstable locomotion. Although the pattern gradually stabilized over several weeks, plantigrade placement of the denervated HL was facilitated only if the non‐denervated HL stepped faster during split‐belt locomotion. In the opposite split‐belt condition, the pattern remained unstable. In the least affected cat, although no abnormal paw placements were observed 1 day post‐denervation, HL locomotion was considerably more stable with the denervated HL stepping faster during split‐belt locomotion. Preliminary results suggest that ankle extensors are important for the spinal control of left‐right coordination and that adaptation can be shaped by split‐belt locomotion.
Astrocytes mediate Short‐term Plasticity of Breathing
Authors: Okada Yasumasa , Takeda Kotaro , Someya Hiroshi , Oku Yoshitaka , Miwakeichi Fumikazu , Ishiguro Makio , Tamura Yoshiyasu , Pokorski Mieczyslaw
Published: June 2021

Respiratory plasticity contributes to stabilization of ventilation. However, its cellular mechanism has not been clarified. We hypothesized that astrocytes mediate respiratory plasticity, based on the discovery that astrocytes are actively involved in neural plasticity in various brain functions. To examine our hypothesis, we analyzed the effects of ONO‐2506, an inhibitor of astrocytic activation, on post‐hypoxic short‐term potentiation of breathing. Ventilatory parameters of unanesthetized adult mice were measured by whole body plethysmography (n=11). To test the hypoxic response, mice breathed room air, then hypoxic gas (7% O2, 93% N2), and again room air. After recording control hypoxic response with recovery time course, low and high doses of ONO‐2506 was ip injected. Although ONO‐2506 little affected ventilatory augmentation during hypoxic loading, it suppressed post‐stimulus potentiation of breathing. High dose ONO‐2506 completely eliminated post‐stimulus potentiation. We conclude that post‐stimulus potentiation of breathing is mediated by astrocytes. We propose a model to explain the mechanism of post‐hypoxic potentiation. Hypoxia stimulates the respiratory neuronal network through the excitation of the carotid body. Neurotransmitters spilled from excited synapses activate nearby astrocytes. These activated astrocytes persistently excite respiratory neuronal network by continuously releasing gliotransmitters, and short‐term respiratory plasticity emerges.
Relation Between Diaphragm EMG and Ventilation in Rats Acclimatized to Hypobaric Sustained Hypoxia
Authors: Moya Esteban , Berruecos Steve , Fu Zengxing , Powell Frank
Published: June 2021

Exposure to chronic sustained hypoxia in healthy subjects, for example during acclimatization to high altitude, increases ventilation in normoxia and increases the ventilatory response to acute hypoxia or hypercapnia. Laboratory rodents have proved to be a useful model for studying the mechanisms of such ventilatory acclimatization to hypoxia. However, it is not known if all respiratory muscles contribute similarly to increases in ventilatory drive and chemoreflexes with acclimatization. Here we tested the hypothesis that the diaphragm contributes to increases in ventilatory responses to hypoxia and hypercapnia equally before and after acclimatization. Therefore, we measured diaphragm electromyogram activity and related the amplitude of the rectified and integrated signal (EMGdia) to tidal volume (Vt) in rats exposed to one week of sustained hypoxia. We made an abdominal incision in male Sprague‐Dawley rats and implanted a two‐electrode transmitter to telemeter EMG activity from the diaphragm muscle. After recovery, we simultaneously measured ventilation with barometric pressure plethysmography and EMGdia with telemetry in awake, unrestrained rats before and after 7 days of exposure to hypobaric hypoxia (PO2 = 80 Torr). EMGdia positively correlated with Vt when breathing was stimulated by hypoxia, hypercapnia or both, and this relationship was not changed after chronic hypoxia. This shows that ventilatory acclimatization to hypoxia does not change the contribution of the diaphragm to ventilatory drive and chemoreflexes.
Supported by NIH RO1 HL‐081823.
Phrenic Long‐term Facilitation (pLTF) Following Acute Intermittent Hypoxia is NADPH Oxidase Independent in a Rat Model (SOD1G93A) of ALS
Authors: Nichols Nicole , Mitchell Gordon
Published: June 2021

Acute intermittent hypoxia (AIH; 3, 5‐min episodes, 11% O2) elicits a form of respiratory plasticity known as pLTF. In normal rats, pLTF requires NADPH oxidase activity and superoxide formation. Since enhanced pLTF is observed in SOD1G93A rats at disease end‐stage (Nichols et al., 2011) despite increased superoxide dismutase‐1 expression and decreased superoxide accumulation in phrenic motor neurons (Satriotomo et al., 2008), we tested the hypothesis that AIH‐induced pLTF is NADPH oxidase independent in anesthetized,paralyzed and ventilated end‐stage SOD1G93A rats (versus age‐matched, wild‐type littermates). Following intrathecal injections of vehicle (control) or an NADPH oxidase inhibitor (apocynin; 600μM; 12μL), pLTF was abolished in wild‐type (7±3%, n = 4; controls: 40±4%, n=5; p<0.05), but not end‐stage SOD1G93A rats (78±15%, n = 4; controls: 92±18%, n=4; p>0.05). Since pLTF is no longer dependent on NADPH oxidase activity in end‐stage SOD1G93A rats, we suggest that key molecular targets of reactive oxygen species that normally constrain pLTF (e.g. protein phosphatases) are down‐regulated with disease progression in ALS.

[Supported by NIH K99 HL119606, R01 HL080209 and R01 HL069064]
Stretch Activation of Angiotensin II Type 1 Receptor Contributes to Ventilator‐induced Diaphragm Dysfunction
Authors: Hall Stephanie , Morton Aaron , Smuder Ashley , Wiggs Michael , Sollanek Kurt , Powers Scott
Published: June 2021

Mechanical ventilation (MV) is a life‐saving intervention for patients in respiratory failure. Unfortunately, prolonged MV results in diaphragmatic atrophy and contractile dysfunction (collectively termed ventilator‐induced diaphragm dysfunction, VIDD). The development of VIDD is important because diaphragm weakness can contribute to problems in weaning patients from MV. Currently, no standard treatment exists to prevent VIDD and understanding the causation of VIDD is vital for the development of a therapeutic intervention. Recent evidence reveals that increased plasma levels of Angiotensin II (Ang II) promotes oxidative stress and fiber atrophy in limb muscles. Our preliminary experiments suggest that Ang II type 1 receptor (AT1R) activation contributes to VIDD but MV‐induced AT1R activation in the diaphragm is independent of circulating Ang II levels. Therefore, using animal rat model of MV, we tested the hypothesis that MV‐induced stretch activation of AT1R in the diaphragm is required for the development VIDD. Two structurally different AT1R antagonists were administered during 12 hours of MV. Olmesartan has an ideal chemical structure to inhibit stretch‐induced AT1R activation whereas irbesartan does not prevent stretch‐induced AT1R activation. Both drugs are effective at blocking Ang II binding to AT1R. Treatment of animals with olmesartan protected against VIDD whereas irbesartan did not protect against VIDD. These results suggest that stretch activation of AT1R is essential for the development of VIDD. Importantly, these experiments provide evidence that the FDA approved drug olmesartan may have clinical benefits in the protection against VIDD in humans.
A Novel Approach to Target Motoneurons Using Mesoporous Silica Nanoparticles
Authors: Gonzalez Maria , Brinker Jeffrey , Sieck Gary , Mantilla Carlos
Published: June 2021

Diseases of the central nervous system commonly affect motoneurons. In particular, spinal cord injury, aging and neurological disorders can cause motoneuron dysfunction or loss. There are no effective diagnostic tools or treatments that specifically target motoneurons. We hypothesized that nanoparticles can be used to target drug delivery to motoneurons. Specifically, mesoporous silica nanoparticles (MSNPs) may be ideal since they can be independently modified allowing loading of various cargos and surface‐engineered for bio‐functionality. The aim of this study was to characterize the role of nanoparticle size, concentration, time of incubation and surface coating on uptake by cultured motoneurons. Four different fluorescently‐labeled MSNPs were evaluated: PEG‐PEI‐Ace MSNPs of 150 nm and 50 nm, lipid coated MSNPs (protocell ‐ 140 nm) and PEG‐PEI MSNPs (50nm). PEG‐PEI‐Ace showed significant greater motoneuron uptake (p<0.05) after longer periods of incubation (12h for 50 nm & 24h for 150 nm). Protocells and PEG‐PEI achieved maximal uptake by 3h. Differentiated motoneurons displayed significant uptake at cell soma and dendrites. Regardless of cell density and duration of treatment, there was minimal evidence of cytotoxicity by any MSNP. In conclusion, MSNPs can be used to target delivery to motoneurons. Specifically, coating, size and concentration affect motoneuron uptake. Smaller and coated PEG‐PEI‐Ace MSNPs showed substantial uptake by motoneurons. These results may help inform the appropriate characteristics of MSNPs for specific in vivo application.
Acute caspase inhibition attenuates progressive vascular remodeling in established Pulmonary Arterial Hypertension
Authors: Happé C M , Szulcek R. , Rol N. , Schalij I. , Westerhof N. , Vonk‐Noordegraaf A , De Man F.S. , van Nieuwe Amerongen G.P. , Bogaard H.J.
Published: June 2021

In pulmonary arterial hypertension (PAH) a phenotypic switch of the endothelium results in exuberant cell growth and vascular remodeling. Recent animal models of PAH show a high cell turnover with coexisting high rates of apoptosis and proliferation. In vitro experiments suggest that high shear stress induces a loss of PAH patient derived microvascular endothelial cells (MVEC). We tested whether an impaired response to increased shear stress and ongoing apoptosis are involved in vascular remodeling in PAH.
Rats with progressive PH were treated for 2 weeks with the pan‐caspase inhibitor Z‐Asp. PAH patient derived MVECs were treated with Z‐Asp and subjected to increased levels of fluid shear stress to simulate the hemodynamics of the PAH lung.
Analysis revealed a significant decrease in cleaved caspase 3 positive cells in lung endothelial cells (0.6±0.1 vs. 0.2±0.1, p=0.02) and smooth muscle cells (2.2±0.2 vs. 1.2±0.5, p=0.02). A reduction in pulmonary vascular resistance (0.9±0.2 vs. 0.5±0.1, p=0.01) with Z‐asp was associated with a decrease in intima thickness (24±4 vs. 14±2, p=0.001). Detailed analysis showed a decrease in the proliferative marker PCNA in endothelial cells (1.5±.4 vs. 0.8±0.2, p=0.05). Mechanistically, Z‐Asp in MVECs improved shear adaptation and prevented shear‐induced cell loss by restoring protein surface levels of the mechanosensor CD31.
In conclusion, we showed that caspase inhibition in experimental PAH attenuates vascular remodeling by restoration of endothelial shear adaptation and apoptosis reduction. Our data suggests that new PAH treatment concepts should consider apoptosis prevention instead of induction.
Aquaporin 1 (AQP1) Enhances Pulmonary Arterial Smooth Muscle Cell (PASMC) Migration and Proliferation Via Regulation of β‐Catenin
Authors: Yun Xin , Jiang Haiyang , Lai Ning , Shimoda Larissa
Published: June 2021

PASMC proliferation and migration are key components in the vascular remodeling that occurs during development of hypoxic pulmonary hypertension. We previously showed that hypoxia increased expression of AQP1, a water channel, and that AQP1 regulates PASMC proliferation and migration via a mechanism that requires the C‐terminal tail but is independent of water transport. β‐catenin is an important regulator of cell migration and proliferation, and may interact with AQP1 in certain cell types. In this study, we wanted to elucidate the mechanism by which AQP1 regulates PASMC proliferation and migration by exploring whether AQP1 and β‐catenin interact in PASMCs, and if so, determine whether the AQP1 C‐terminal tail was required. Rat PASMCs were harvested from distal pulmonary arteries, plated onto culture dishes and infected with adenoviral constructs expressing green fluorescent protein (AdGFP), as a control for infection, wild‐type AQP1 (AdWT) or AQP1 lacking the C‐terminal tail (AdCT). Compared to AdGFP cells, infection with AdWT, but not AdCT, increased PASMC migration, proliferation and β‐catenin protein expression. Knockdown of β‐cateninin PASMCs infected with AdWT prevented the AQP1‐induced increase in proliferation and migration. Our results suggest that increased AQP1 levels upregulate β‐catenin protein levels via a mechanism requiring the AQP1 C‐terminal tail, enhancing PASMC proliferation and migration.
The Functional Coupling of CaSR and TRPC6 Contributes to Enhanced Proliferation in Pulmonary Arterial Smooth Muscle Cells
Authors: Fernandez Ruby , Jimenez‐Perez Laura , Song Shanshan , Tang Haiyang , Yuan Jason
Published: June 2021

An increase in cytosolic free Ca2+ ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) is not only a major trigger for pulmonary vasoconstriction but also an important stimulus for PASMC proliferation.We have previously shown both extracellular Ca2+‐sensing receptor (CaSR), a G protein‐coupled receptor, and TRPC6, a transient receptor potential cation channel, are upregulated in PASMC from idiopathic pulmonary arterial hypertension (IPAH) patients. Furthermore, extracellular Ca2+‐induced increase in [Ca2+]cyt was enhanced in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH) compared to normal patients. However, the implication of increased CaSR and TRPC6 in pulmonary hypertension remains unknown. In this study, our data show the inhibition of CaSR by siRNA significantly inhibited hypoxia‐induced proliferation in both normal and IPAH PASMC. Furthermore, IPAH‐PASMC treated with siRNA targeting CaSR or TRPC6 inhibited Ca2+‐induced Ca2+ entry, suggesting functional coupling of CaSR and TRPC6. In addition, PDGF‐induced proliferation in IPAH‐PASMC was inhibited by blocking either CaSR or TRCP6 via inhibitors NPS2143 and 2‐APB, respectfully, while NPS2143 was unable to block PDGF‐induced proliferation in normal PASMC. The data from this study indicate that upregulation of CaSR is functionally coupled to TRPC6 channels to increase [Ca2+]cyt; the enhanced extracellular Ca2+‐induced increase in [Ca2+]cyt due to upregulation of CaSR and TRPC6 plays an important role in PASMC proliferation. This suggests that CaSR could be a novel therapeutic candidate for the treatment of PH.
The Chaperone Protein Hsp90 is Upregulated in Human Pulmonary Hypertension
Authors: Ruffenach Gregoire , Le Guen Marie , Chabot Sophie , Breuils‐Bonnet Sandra , Tremblay Eve , Provencher Steeve , Bonnet Sebastien
Published: June 2021

INTRODUCTION

Human and animal pulmonary arterial hypertension (PAH) is characterized by a selective activation of several transcription factors including STAT3, NFAT and HIF‐1α, which regulates pulmonary artery smooth muscle cells (PASMC) proliferation and apoptosis. The activation mechanisms of these factors in PAH remain elusive. Hsp90 is a major molecular chaperone that is plays a pivotal role in assisting correct folding and functionality of its client proteins. The Hsp90 client proteins include a wide variety of transcription factors like NFAT, STAT3 and HIF‐1α. Recent evidences have shown that miR‐223 may regulates HsP90 expression in cancer, which share many similarities with PAH. We thus hypothesized that the miR‐223/Hsp90 axis is aberrantly expressed in PAH patients, contributing to PAH‐PASMC phenotype.

METHODS and RESULTS

Using immunoblot, we showed that Hsp90 is upregulated in lungs, and PASMC of PAH patients compared to non‐PAH donors. This upregulation in Hsp90 is associated with a significant downregulation of miR223 in PAH; upregulation of HIF‐1α, NFAT and STAT3 activation measured by immunoblot and nuclear translocation assay. In primary cultured PASMC from 3 PAH and 3 control patients, we demonstrated that miR‐223 upregulation using mimic or Hsp90 molecular inhibition using siRNAs significantly reduces PAH‐PASMC proliferation and resistance to apoptosis. Finally, using monocrotaline‐induced PAH, we showed that miR223 nebulization decreases total pulmonary resistance by decreasing distal PA wall thickness.

CONCLUSION

The miR223/Hsp90 may represent a new signal hub in PAH, accounting for the activation of several transcription factors contributing to PAH‐PASMC phenotype and thus vascular remolding seen in PAH.
Human Mannose‐Binding Lectin (MBL2) Inhibitor Prevents Renal Injury in a Novel Animal Model of Endemic Hemolytic Uremic Syndrome
Authors: Ozaki Masayuki , Liu Bohan , Kushak Rafail , Rosales Ivy , Kang Yulin , Pavlov Vasile , Grabowski Eric , Stahl Gregory
Published: June 2021

Endemic hemolytic uremic syndrome (eHUS) is a worldwide problem and its pathophysiology is not fully elucidated. We hypothesized that mannose‐binding lectin (MBL2), an initiating factor of lectin complement pathway activation, plays a crucial role in eHUS. We generated a novel human MBL2 expressing mouse (MBL2 KI) that lacks murine MBLs (MBL2+/+Mbl1‐/‐Mbl2‐/‐). The novel eHUS model was designed with MBL2 KI mice injected (day 0) with shiga toxin‐2 (Stx‐2; 125 pg/g, ip) with or without anti‐MBL2 antibody (3F8; 30 µg/g, ip). Blood cell counts were obtained and creatinine (Cr), blood urea nitrogen (BUN), Cystatin C, and MBL2 sera levels were measured on day 4. Hematoxylin/eosin and immunohistochemical staining with anti‐β‐fibrin antibody were performed on renal sections. Stx‐2 induced weight loss, anemia, thrombocytopenia and increased sera Cr, BUN, and Cystatin C levels compared to control mice. Treatment with 3F8 significantly attenuated Stx‐2 induced thrombocytopenia, MBL2, Cr and Cystatin C levels. Staining demonstrated detached tubular epithelial cells and β‐fibrin deposition in glomeruli in Stx‐2 treated mice. Inhibition of MBL2 with 3F8 prevented Stx‐2 induced renal injury and β‐fibrin deposition. In conclusion, a relevant eHUS model using MBL2 KI mice and Stx‐2 was developed. MBL2 inhibition significantly protected against thrombocytopenia and renal injury in eHUS. This novel eHUS model can be used to study the role of complement, particularly the lectin pathway. The data suggest an important role of MBL2 in eHUS. Funded by AI089781 and HL056086
Strain‐dependent metabolic phenotype responses to various exercise paradigms in inbred mice
Authors: Avila Joshua , Kim Seung , Massett Michael
Published: June 2021

The aim of this study was to determine the interaction between genetic background and training volume (intensity x duration) on exercise performance and myogenic proteins. Male mice from four inbred strains: SJL/J, C57BL/6J, NON/ShiLtJ, and 129S1/SvlmJ completed a graded exercise test before and after four or eight weeks of treadmill training. Mice from each strain were assigned to one of three groups (n=6/group): sedentary control (Sed), continuous running (Low) (65% of max) and high‐intensity interval training (HIIT) (6 sets of 8 min at 85% of max for 8 min, followed by 2 min of low intensity at 50% of max for 2 min). Changes in exercise time were used to assess performance. Protein content of PGC‐1α, SIRT1, Glut4, Cytochrome C, Hexokinase, TFAM and NRF was examined in gastrocnemius muscle to assess the metabolic adaptation to the different intensities of exercise training. For change in time, there were significant main effects for strain, intensity and duration. There was a significant strain effect for protein content for all proteins, except PGC‐1α. Duration had a significant effect for all proteins, with protein content being greater at 8 weeks than 4 weeks. A significant intensity effect was observed for PGC‐1α and Cytochrome C with Low and HIIT being significantly greater than Sed. These data indicate that genetic background and training duration have a strong influence on adaptations to training in skeletal muscle protein. Adaptations to training in skeletal muscle proteins are not directly related to changes in exercise capacity.

Supported by NIH grant HL085918 to MPM, Sydney and JL Huffines Institute for Sports Medicine and Human Performance to JJA, William Townsend Porter fellowship from the APS to JJA.
GABA A Receptor Gamma 3 is a New Candidate Gene for Cardiovascular and Behavioral Disorders
Authors: XIA JIXIANG , Batchu Sri , Donlon Kathy , Murashev Arkady , Korshunov Vyacheslav
Published: June 2021

We previously discovered a significant locus on mouse chromosome (chr) 7 that controls elevated heart rate in a genetic cross between C3HeB and SJL mice. Association mapping within the locus revealed three subunits of gamma‐aminobutyric acid (GABA) A receptors. The mouse chr7 locus is syntenic to human chr15q locus and is important for mental disorders as well as heart rate variation. The goal of this study was to further investigate the GABAA receptors as candidate genes in the regulation of the cardiovascular system. First, we evaluated expression profiles of the candidate genes in several organs between C3HeB and SJL strains. There was higher mRNA expression of all three GABAA receptors in mouse brain compared to other organs in both strains. We found that alpha5 (~14‐fold) and gamma3 (~5‐fold) were significantly higher in brains from C3HeB compared to SJL mice. In addition, gamma3 mRNA levels were greater in the C3HeB hearts (~20‐fold). Western blot analyses of brain and heart confirmed higher expression of the gamma3 protein in C3HeB versus SJL mice. We genetically targeted the gamma3 gene by using a knockout first and promoter driven reporter tag strategy. Mice with positive alleles for gamma3 showed persistent beta‐galactosidase staining in the brain at embryonic days E12.5 and E15.5. In summary, GABAA gamma3 receptor is a strong candidate gene for elevated heart rate within the mouse chr7 locus. We developed a new mouse model with conditional potential that will allow us to investigate contribution of GABAA gamma3 receptor in cardiovascular and behavioral disorders.
Searching for the genetic basis and mechanism of nephrogenesis defects in the HSRA congenital solitary kidney rat
Authors: Wang Xuexiang , Johnson Ashley , Guo Zibiao , Jia Zhen , Garrett Michael
Published: June 2021

Our lab recently developed a unique rat model (HSRA rat) with 50‐75% of offspring developing with a single kidney. The HSRA model exhibits ~20% less nephrons in the remaining kidney (in addition to the loss of one kidney), early glomerular and kidney hypertrophy, and elevated single nephron GFR, leading to progressive kidney injury and decline in kidney function. The HSRA model is also highly susceptible to hypertension induced kidney injury. With a detailed understanding of the physiological mechanism of long‐term cardiovascular and renal hemodynamics, we now seek to establish the mechanism of nephron deficiency in the solitary kidney as well as identify the exact genetic basis leading to the failure of one kidney to develop. RNA sequencing of embryonic kidney isolated at key stages of nephrogenesis (E14.5, 15.5, and 16.5) between embryos exhibiting a single kidney and two‐kidney control identified genes/pathways that may explain decreased nephrons exhibited in the solitary kidney. A recent genome‐wide association study performed by others using the heterogeneous stock (HS) rat (progenitor of HSRA model) identified genomic loci on chromosome 4, 10, and 14 associated with the solitary kidney. Whole genome sequencing of the HSRA model is currently planned, which when combined with identified loci, will expedite identification of genes contributing to solitary kidney. In summary, the identification of the specific gene defect, modifier genes, and signaling pathways that lead to loss of one kidney and nephron deficiency in the remaining kidney will provide a mechanistic link between solitary kidney genotype and the development of kidney dysfunction exhibited by the HSRA.
Cell‐Specific Regulation of BK Channel and WNK Kinases in Cortical Collecting Duct (CCD) by Dietary K
Authors: Carrisoza Rolando , Subramanya Arohan , Carattino Marcelo , Kleyman Thomas , Satlin Lisa
Published: June 2021

WNK kinases regulate Na absorption and K secretion in the distal nephron (DN) in response to dietary K intake. Evidence now suggests that WNKs regulate apical BK channel expression; specifically, WNK4 inhibits BKα by targeting the channel for degradation through an ubiquitin‐dependent pathway. Although WNK1 and WNK4 show distinct subcellular distribution patterns, cell‐specific regulation by dietary K has not been studied. Thus, CCDs from NZW rabbits fed a high (HK) or low (LK) K diet for 10 d were fixed, perfused with antibodies (Abs) against BKα, L‐WNK1 (ex. 1) or WNK4 (+ fluorescent 2o Abs), or rhodamine‐peanut lectin (PNA), which binds to apical surfaces of intercalated (IC) but not principal (PC) cells, and examined by confocal microscopy. In LK CCDs, BKα localized predominantly to cilia with minimal detection along the apical membranes of ciliated > non‐ciliated cells. In HK CCDs, apical BKα was enhanced in non‐ciliated > ciliated cells. WNK1 was not detected in LK CCDs (2) but was observed in HK CCDs (4) in a basolateral (BL) distribution in all cells and robustly along the apical membrane of PNA+ cells. In contrast, cytoplasmic and BL WNK4 was observed in all cells in LK CCDs (2) but was minimally expressed in a BL distribution in non‐ciliated cells in HK CCDs (2). In sum, adaptation to a HK diet includes an increase in apical BKα and L‐WNK1 and reduction in WNK4 expression in ICs, suggesting that increased L‐WNK1 and reduced WNK4 contribute to enhanced BK channel‐mediated K secretion in HK animals. In PCs, HK diet increases apical but not cilia BKα, a response associated with BL WNK1 but absent WNK4. These results are consistent with cell‐specific regulation of BKα and WNK kinase expression by dietary K in the DN.
Familial Hyperkalemic Hypertension (FHHt) Proteins Form an Endocytic Switch for the Renal Excretory Channel, KCNJ1
Authors: Welling Paul , Fang Liang , Kim Boyoung , Wade James , Woodward Owen
Published: June 2021

The kidney potassium channel, ROMK (KCNJ1), controls urinary potassium excretion, in states of dietary potassium deficiency the channel is down‐regulated by the endocytic clathrin adaptor protein, ARH. Inappropriate ROMK endocytosis contributes to hyperkalemia in Familial Hyperkalemic Hypertension (FHHt), a rare disorder caused by mutations in WNK 1, CUL3, and KLHL3. Here we explore how WNK1 and the E3 ligase complex containing CUL3 / KLHL3 regulate ARH ubiquitination, abundance, and ROMK expression. We found ARH directly binds to and is ubiquitinated by the CUL3/ KLHL3 E3 ligase complex, leading to ARH degradation. WNK1 blocks ARH degradation and KLHL3 binding by controlling phosphorylation of ARH at serine 14. Phosphorylation of ARH led to increased abundance as did the S14D phosphomimetic by blocking the direct ARH‐KLHL3 interaction. Further we found WNK1 activates a kinase cascade resulting in the direct phosphoryation of ARH S‐14 by the ERK5 kinase, as demonstrated by in vitro phosphorylation assays with recombinant ERK5 and in cells with the ERK5 inhibitor BIX02189, a finding consistent with the observed increase in activated ERK5 and ARH in mice fed a low K+ diet. These studies provide a molecular insight to understand how mutations in WNK1 and CUL3‐KLHL3 acerbate channel endocytosis and disrupt potassium homeostasis in FHHt.
5/6 Nephrectomy Induces Changes in Immune and Inflammatory Gene Expression in the Left Ventricle
Authors: Kriegel Alison , Casati Marc , Liu Pengyuan , Liang Mingyu
Published: June 2021

Chronic kidney disease (CKD) patients have increased cardiovascular risk. We have used 5/6 nephrectomy (5/6 NX) in Sprague Dawley rats to investigate the molecular mediators of cardiac pathology in this condition. Understanding gene expression changes in the heart could yield valuable information about pathology mediators and identify therapeutic targets. The goal of this study was to characterize changes in left ventricle (LV) gene expression following 5/6 NX. Reproducible alterations in LV structure and function were observed in the weeks following 5/6 NX including enhanced function (2 weeks), hypertrophic remodeling (4 and 5 weeks), and dilation with reduced function (7 weeks post). We performed next‐generation sequencing of mRNA extracted from the LV of 5/6 NX and sham rats at these time points (2 pools/group, 2‐3 animals/pool). Genes from our data set with an average fold change of >1.5 (vs. sham) were analyzed using DAVID and Ingenuity Pathway Analysis. Several signaling pathways were enriched with identified genes in a time dependent manner, corresponding with pathological LV changes. However, we found enrichment of altered genes in numerous immune system and inflammatory pathways throughout the study, including pathways related to atherosclerosis, granulocyte and agranulocyte movement, and innate and adaptive immune signaling. This study provides the first gene expression data showing that a reduction in renal mass alone can drive broad immune related gene expression changes in LV tissue. Further, these genes are altered beginning as early as 2 weeks after 5/6 NX, long before cardiac remodeling or dysfunction are observed. Support 13SDG17100095 and 8UL1TR000055.
Demonstrating a Role for Nuclear APC in Intestinal Cellular Differentiation
Authors: Miller Matthew , Neufeld Kristi
Published: June 2021

Colon cancer is the second leading cause of cancer‐related mortality in the United States, resulting in over 50,000 deaths annually. Approximately 80% of colon cancers begin with a mutation in the gene coding for the tumor suppressor, Adenomatous Polyposis Coli, APC. Facilitated by two nuclear localization signals (NLS) and multiple nuclear export signals, APC protein can shuttle between the cytoplasm and the nucleus of cells. To understand the functions of nuclear APC, our lab generated a knock‐in mouse model with mutations introduced that inactivate both Apc NLS, thus dramatically decreasing their amount of nuclear Apc. We have previously shown increased proliferation and Wnt target gene expression in intestinal epithelial cells from ApcmNLS/mNLS mice, suggesting a role for nuclear APC in inhibition of proliferation and Wnt signaling. Here we examine the role of nuclear Apc in intestinal epithelial differentiation and stem cell homeostasis. Paraffin‐embedded tissue from the intestines of Apc+/+ and ApcmNLS/mNLS mice was sectioned and stained for markers of enterocytes (alkaline phosphatase), goblet cells (alcian blue), enteroendocrine cells (chromogranin A) and Paneth cells (lysozyme C). Quantification of positive cells indicated that ApcmNLS/mNLS mice had fewer differentiated enterocytes, goblet cells, and Paneth cells, yet more enteroendocrine cells than their wild‐type littermates. Together, these data support a role for nuclear Apc in promoting enterocyte, goblet cell, and Paneth cell differentiation while inhibiting enteroendocrine cell differentiation. No significant change was observed in quiescent stem cell (DCAMKL‐1) populations. Information gathered from this analysis will contribute to our understanding of the mechanism of intestinal differentiation and tumorigenesis.
Heme Oxygenase‐1 Deficiency Promotes Embryonic Stem Cell Differentiation
Authors: Yet Shaw‐Fang
Published: June 2021

Pluripotent embryonic stem cells (ESCs) are promising donor sources in cell therapies for various diseases. Many signals including cytokines, growth factors, and oxidative stress influence ESC survival and differentiation. The stress response protein heme oxygenase‐1 (HO‐1) has been shown to protect tissues from injury via its antioxidative and anti‐inflammatory functions. Given that ESCs are highly proficient in antioxidant defense, we hypothesized that HO‐1 might contribute to ESCs' antioxidant defense and differentiation. To test this hypothesis, we isolated HO‐1‐deficient (HO‐1–/–) ESCs from mouse blastocysts. As with D3 wild type ESCs, HO‐1–/– ESCs exhibit high levels of pluripotent markers Oct3/4 and SSEA‐1, strong alkaline phosphatase activity, and low levels of reactive oxygen species (ROS). Since elevated levels of ROS can initiate differentiation, we used an embryoid body (EB) model and harvested EBs at day 3, 5, and 7 to first examine ROS levels. ROS levels increased during ESC differentiation, reaching maximum levels at day 5. However, HO‐1 deficiency enhanced ROS levels at day 3. We next examined differentiation marker gene expression by quantitative RT‐PCR. The endoderm marker GATA6 increased during the course of differentiation; interestingly, HO‐1 deficiency enhanced GATA6 levels by 9‐fold at day 3. Furthermore, the mesoderm marker Brachyury was 50‐fold higher than D3 at day 3. However, no difference was observed in ectoderm marker FGF5 between D3 and HO‐1–/– ESCs at day 3. Collectively, these results suggest that HO‐1 might modulate ESC differentiation, particularly endoderm and mesoderm, via regulating cellular ROS levels. This work was supported in part by National Health Research Institutes (Taiwan) grants CS‐103‐PP‐05 and 03A1‐CSSP‐01.