bluuebunny/crossref_metadata_2025 huggingface dataset:
Intermittent Hypoxia Upregulates Angiotensin II Generating Pathways in Human Preadipocytes
Authors: Becari Christiane , Singh Prachi , Polonis Katarzyna , Somers Virend K.
Published: November 2023

Background

Obstructive sleep apnea (OSA) is characterized by repetitive intermittent hypoxia and reoxygenation during sleep, and is a risk factor for cardiometabolic diseases including hypertension and diabetes. The renin‐angiotensin system (RAS) is an important cardiovascular and metabolic regulator playing key roles via angiotensin peptides. Recent studies demonstrate that all components of the RAS, including enzymes responsible for AngII generation, such as angiotensin converting enzyme (ACE) and chymase, are present in adipose tissue. Furthermore adipose tissue significantly contributes to systemic levels of AngII, thereby potentiating development of hypertension in obesity. Given that OSA patients are mostly obese, the localized effects of RAS in adipose tissue are likely an important mediator of OSA‐related hypertension. This study sought to investigate the effects of intermittent hypoxia (IH), a hallmark of OSA, on Ang II generation pathways in preadipocytes.

Methods/Results

RAS plays an important role in adipose tissue via its action on preadipocytes. Therefore, we examined the effects of IH in primary cultures of human white preadipocytes (HWP). HWP cells were exposed to repeated cycles of 30 min‐21% O2 (normoxic) and 30 min‐1% O2 (hypoxic) at 37°C for 24 hours. Cells grown in continuous normoxic conditions were used as controls. mRNA and protein expression of ACE, chymase, renin, AT1, and AT2 receptors were investigated by semi‐quantitative RT‐PCR and Western blot, respectively. Ang II secretion was examined by enzyme‐linked immunosorbent assay in condition medium.

Results

IH upregulated ACE (P=0.04) and chymase (P=0.05) expression while the expression of renin (P=0.94), AT1R (P=0.30), and AT2R (P=0.58) receptors did not change in HWP. Additionally, IH increased Ang II secretion in conditioned media (P=0.01).

Conclusions

This is the first study to demonstrate intermittent hypoxia‐mediated upregulation of Ang II generating enzymes ACE and chymase, along with increased AngII secretion in HWP. Together, our data strongly support the role of IH in upregulating RAS via increases in AngII. These studies suggest a role for adipose tissue and RAS in OSA related pathophysiology.

Support or Funding Information

NIH R01 grant (#HL65176); Becari C. is supported by grant from CNPq‐Brazil (# 203802/2014‐4).
Proinsulin C‐peptide prevents VEGF release from ARPE‐19 cells: A novel signaling mechanism
Authors: Kolar Grant R. , Yosten Gina L. C.
Published: November 2023

Diabetic macular edema is associated with the breakdown of tight junctions between retinal pigment epithelial (RPE) cells, which form the blood‐retinal barrier and provide metabolic support to the photoreceptors. Loss of tight junctions within this single‐celled layer of the retina leads to enhanced permeability of the blood‐retinal barrier, intra‐ocular swelling, and macular edema. The mechanisms underlying RPE dysfunction in diabetic macular edema are not fully understood, but excessive VEGF secretion and signaling appears to play a role. Because proinsulin C‐peptide has been shown to prevent and reverse diabetic eye disease and protect against the deleterious effects of VEGF hyper‐secretion, we evaluated the effect of C‐peptide on VEGF release from the human RPE cell line, ARPE‐19. Exposure of ARPE‐19 cells to C‐peptide inhibited basal and high glucose‐stimulated VEGF release. Furthermore, C‐peptide suppressed insulin‐stimulated VEGF release from these cells as well. Because C‐peptide has been shown to regulate activity of the Na+/K+ ATPase in multiple cell types, we hypothesized that C‐peptide prevents the hyper‐secretion of VEGF induced by high glucose by enhancing Na+/K+ ATPase activity. However, treatment with the Na+/K+ ATPase inhibitor ouabain also decreased VEGF release from ARPE‐19 cells exposed to high glucose. We therefore propose that C‐peptide prevents excessive VEGF release from the retinal pigment epithelium by suppressing Na+/K+ ATPase activity, which could have important implications for the management of diabetic macular edema.
The Effects of Hyperglycemia on Insulin Resistance in a Drosophila model of Alzheimer's disease
Authors: Cline Delaena , Paddock Brie
Published: November 2023

Alzheimer's disease (AD) and Type‐2‐Diabetes Myelitis (T2DM) are two increasingly prevalent diseases throughout the world, with evidence suggesting T2DM is a risk factor for AD. Alzheimer's disease, a neurodegenerative disorder, is characterized by the accumulation of A‐β leading to increased loss of memory and decreased synaptic plasticity. Hyperglycemia, associated with T2DM, leads to an exacerbation of amyloid accumulation and deposition within the brain. This deposition increases the likelihood of developing AD in these individuals and worsens present symptoms of those already suffering, including memory loss. It is unknown if hyperglycemia precedes amyloid‐induced pathogenesis or if accumulation of amyloid protein results in abnormal glucose handling. This study seeks to clarify this uncertainty by testing how high levels of A‐β within the nervous system of Drosophila melanogaster will affect the use of insulin in processing glucose. Using a standard Oral Glucose Tolerance Test, we are measuring the ability of this AD model to process a glucose challenge after 16 hours of starvation. Our current data indicates higher levels of circulating glucose in the AD model. These data suggest that coexpression of human amyloidogenic proteins in the nervous system may result in glucose handling abnormalities similar to those of T2DM.

Support or Funding Information

College of Arts and Sciences, Arcadia University
Mitogen‐inducible Gene 6 Accelerates the Progression to Diabetes by Inhibiting EGFR‐Mediated Repair Mechanisms
Authors: Fong Kimberley M , Fueger Patrick T
Published: November 2023

Type 1 diabetes (T1D) is caused by autoimmune‐mediated beta cell destruction. Following beta cell injury, the pancreas initiates a cellular repair or regeneration program, which includes epidermal growth factor receptor (EGFR) signaling. However, upon irreparable beta cell damage, EGFR signaling is dampened, disrupting attempts to restore functional beta cell mass and maintain normoglycemia. We have previously demonstrated that the negative feedback inhibitor of EGFR, Mitogen‐inducible gene 6 (Mig6), is induced by the pro‐inflammatory cytokines central to the autoimmune‐mediated beta cell destruction. We also established that pro‐inflammatory cytokines suppress EGFR activation, and siRNA‐mediated suppression of Mig6 restores EGFR signaling. Thus, we hypothesized that a loss of pancreatic Mig6 would protect mice from a chemically‐induced form of diabetes by enhancing EGFR signaling and promoting cellular repair. To this end, we treated mice lacking pancreatic Mig6 (PKO) and their wild‐type littermates (WT) with multiple low doses of streptozotocin (STZ) to induce beta cell death and diabetes or with saline as a control. Whereas STZ‐treated WT mice became hyperglycemic and had reduced beta cell mass, STZ‐treated PKO mice remained euglycemic and glucose tolerant, due to preserved beta cell mass. In addition, nitric oxide (NO) synthase inhibition blocks both the induction of Mig6 and cytokine‐impaired EGFR signaling, and treatment with an NO donor alone induces Mig6. Thus, we conclude that Mig6 ablation promotes beta cell damage repair, hence abating the progression to T1D. Our work suggests that Mig6 may be a novel therapeutic target for preserving beta cell mass in T1D.

Support or Funding Information

This work was supported by NIH grant DK099311 (PTF)
Regional Differences of Pancreatic Islet Area and Beta Cell Density in Obese and Lean Zucker Rats
Authors: Goudy Gabriel , Asano Shinichi , Hoskinson Hannah , Dawson Mariah , Breit Matthew , Olfert Mark , Frisbee Jefferson C , Chantler Paul D , Alway Stephan , Bryner Randall
Published: November 2023

Several studies have characterized the density of beta‐cell mass in rodents reporting greater amounts in the body and tail (which is most proximal to the spleen) compared to the head of the pancreas (located in curved of the duodenum). Whether or not obesity alters the distributions within the pancreas is not known.

PURPOSE

The objective of this study was to evaluate islet area and beta‐cell density in a rat model of obesity and metabolic syndrome.

METHODS

Obese (OZR, n=8) and lean Zucker rats (LZR, n=8) were euthanized at 17‐weeks of age. Pancreases were harvested, sectioned into three regions (head, body, tail) and flash frozen in liquid N2 cooled isopentane. Frozen 7μm thick sections of pancreas tissues were cut, mounted on glass slides, and incubated with an insulin antibody that was labeled with Alexa‐Fluor 546 to identify the beta cells. The tissue was covered in DAPI to label the cell nuclei. Digital images of the pancreas cells were obtained by a Zeiss LSM 510 Confocal microscope using Zenn 2009 software. The insulin positive area of the stained islets was measured using Image J. The images of stained cells and islets were merged and the number of beta cells per islet recorded. ANOVA was used to compare the insulin positive area (islet area), total beta cell number, and beta cell density (beta cell/μm2 insulin positive area) between obese and lean rats with significance set at P<0.05.

RESULTS

There was a significant main effect of phenotype (obese vs lean) for all three dependent variables (Table 1) with overall values lower in the OZR. There was also a significant interaction for phenotype and insulin positive area (islet area) as well as an interaction for phenotype and total beta cell number, but not for beta cell density. This suggests the biggest difference associated with obesity within the pancreas is in the tail region causing a reduced islet area.

CONCLUSION

These data indicate that obesity can significantly reduce islet area but that its affect is primarily in the tail region of the pancreas. The heterogeneity of islet area is consistent with previous findings among the different regions in the pancreas of the rat. Future research needs to examine what mechanisms are responsible for this regional difference.

Support or Funding Information

NIH

AHA

Mean±SE

Group (n=8)
Mean Islet Area (μm2)
Total Beta Cells
Beta cell/μm2 islet area

OZR
LZR
OZR
LZR
OZR
LZR

Head
6859 ±859
8266 ±266
82±26
115±15
0.0124 ± 0.0008
0.0144 ± 0.0008

Body
7831 ±831
7335 ±335
94±4
98±83
0.0123 ± 0.0010
0.0139 ± 0.0008

Tail
4749 ±651*
9475 ±475
56 ±6
136 ±3
0.0118 ± 0.0007
0.0147 ± 0.0004
Effect of dihydrotestosterone treatment on young and elderly mice skeletal muscle
Authors: Wendowski Oskar
Published: November 2023

Background

Sarcopenia, the progressive decline in skeletal muscle mass and function with age, is a debilitating condition. It leads to inactivity, increased susceptibility to falls and eventually to loss of independent living. Despite this, the causes and mechanism(s) underlying sarcopenia are still poorly understood.

Methods

Small muscle fibre bundles isolated from the extensor digitorum longus (edl; a fast‐twitch muscle) and the soleus (a slow‐twitch muscle) of young, old and elderly mice were used to investigated the effects of age and dihydrotestosterone (DHT) treatment on the expression and function of the sodium‐coupled neutral amino‐ amino acid transporter (SNAT) 2 and the sodium‐independent L‐type amino‐acid transporter (LAT) 2.

Results

At all ages investigated, protein synthesis was ~2 times higher in slow‐twitch fibres than in the fast‐twitch fibres. Ageing led to a decrease in protein synthesis in both fibre types. However, the decline was greater in the fast‐twitch than in the slow‐twitch fibres. Moreover, it was accompanied by a reduction in the expression of both SNAT 2 and LAT2 proteins. The decrease in SNAT 2 protein was greater in the fast‐twitch fibres than in the slow‐twitch fibres. In contrast, ageing had no effect on the expression of SNAT2 and LAT2 mRNA. Neither did it affect the uptake of the non‐metabolisable amino‐acid, α‐methylamino‐isobutyric acid. Treating the muscle fibre bundles with physiological concentrations of DHT significantly increased protein synthesis in the fast‐twitch fibres of both young and elderly mice and that of the slow‐twitch fibres in elderly mice only. At both protein and mRNA levels, 5α‐reductase (SRD) 1 was more abundant in skeletal muscle than 5α‐reductase (SRD) 2. It was expressed mostly in skin, tibialis anterior and soleus muscle and its expression increased significantly with age. Chloroquine treatment abolished the increased protein synthesis caused by DHT treatment.

Conclusion

From these results we suggest that sarcopenia arises from an age‐dependent reduction in protein synthesis caused by lack of anabolic stimulus. We also suggest that muscles such as soleus and the tibialis anterior are resistance to sarcopenia because they are able to synthesis more DHT locally. The mechanism of action involves the recruitment of SNAT2 from an intracellular vesicle pool into functioning membrane transport proteins.
Caveolin‐2 Enhances Longevity and Induces Stress Adaptation In C. elegans
Authors: Mehta Meghna D. , Mehta Ekta D. , Schilling Jan M. , Shi Eileen , Fridolfsson Heidi N. , Roth David M. , Patel Hemal H.
Published: November 2023

Caveolae are plasma membrane invaginations common to many cells. Caveolae are enriched in proteins such as caveolin, which are structural proteins that may be stress adaptive. Mammalian cells express three isoforms of caveolin (Cav‐1, Cav‐2, and Cav‐3), whereas C. elegans express only two (Cav‐1 and Cav‐2) with Cav‐2 serving a common function between mammals and C. elegans. To study the benefits of caveolin proteins in stress adaptation, the C. elegans model system was used due to its simplicity and ability to be easily manipulated. We generated C. elegans overexpressing the Cav‐2 (Cav‐2 OE) gene and assessed impact on longevity, egg laying, and stress adaptation. We hypothesized that the over‐expression of Cav‐2 in C. elegans would allow for healthy aging and induce stress adaptation. Cav‐2 OE worms showed significant increases in lifespan, surviving two days longer than their counterparts (p<0.001, n=180, Logrank Test). Cav‐2 OE worms had increased production of eggs with increased duration of egg laying (laying 10–15 more eggs per worm for two days longer, p<0.0001, n=180, 2‐Way RM ANOVA). In order to examine the ability of C. elegans to adapt to stress, we induced stress by exposing the C. elegans to 2% glucose. Cav‐2 OE worms showed enhanced survival compared to N2 controls surviving two days longer (p<0.0022, n=180, Logrank Test). Taken together our data suggests that Cav‐2 is a major regulator of longevity and stress adaptation in C. elegans and C. elegans may serve as an ideal model system to evaluate the impact of caveolin on human pathophysiology and aging.
Drugs That Inhibit The Sarcoplasmic Reticulum Ca2+ ATPase (SERCA) and Prevention of Breast Cancer Cell Proliferation
Authors: Loulousis Melanie , Krager Stacey L , Darcy Yuanzhao L , Tischkau Shelley A , Copello Julio A
Published: November 2023

Breast cancer is the most common cancer in women and a leading cause of death worldwide. Pharmacological modulation of intracellular Ca2+ homeostasis has been proposed as a potential target for therapeutic intervention for breast cancer. In this regard, thapsigargin and cyclopiazonic acid inhibit the Ca2+ ATPase of sarcoplasmic reticulum (SERCA) that pumps Ca2+ into the endoplasmic reticulum (ER) intracellular Ca2+ stores and display high efficacy for killing breast cancer cells. However, these “classic” SERCA blockers are highly toxic and also kill normal cells, thereby preventing use for anticancer therapy. We have determined that new drugs in the benzothiazepine (BZT) family (K201, CGP37157), as well as agents like pimozide and sipatrigine can inhibit SERCA through a novel Ca2+ dependent mechanism. These agents would not affect Ca2+ sequestration in ER when cytosolic Ca2+ levels are high, but potently inhibit this process when cytosolic Ca2+ approaches resting cell levels, which could help prevent ER overload under pathological conditions (ischemia) and provide cellular protection in normal tissues. Therefore, we tested the effectiveness of these drugs to inhibit growth of two breast cancer cell lines, MCF‐7 and MDA‐231, in culture. Our preliminary studies indicate that K201 inhibits MCF‐7 breast cancer cell growth and synergizes with anti‐cancer drugs to increase their effectiveness in killing these cells; thus, MCF‐7 cells are not “protected” by the decreased ER Ca2+ load provided by the SERCA inhibitors; in fact these drugs make them less viable. Building upon this data, we compared the effects of a “classic” SERCA blocker (thapsigargin) to the Ca2+ dependent SERCA blockers above on MCF‐7 and MDA‐231 breast cancer cells. We determined that all SERCA blockers effectively disrupted the metabolic activity of MCF‐7 cells but were much less effective in MDA‐231. In MCF‐7 cells, SERCA blockers also increased the effects of chemotherapeutic drugs (cisplatin, doxorubicin). The results indicate that targeting SERCA has potential to interfere with cancer cell growth. However, our results also suggest that resistance to chemotherapy in MDA‐231 may relate to these cancer cells becoming less dependent on intracellular Ca2+ signaling. We have also determined some of the structural characteristics that are required for BZT to block SERCA. Finally, we also found that drugs reported to do both, protection of normal cells in ischemia and inhibition of cancer cell proliferation, can also display significant SERCA blocking ability. These agents may serve as potential templates for developing new anti‐cancer drugs that target intracellular Ca2+ homeostasis

Support or Funding Information

APS Frontiers in Physiology Research Fellowship to ML

SIUSOM Team Development Award to SAT and JAC
Phosphorylation of α‐actinin‐4 (ACTN4) at serine 159 mimics the biochemical and cellular effects of human kidney disease mutations
Authors: Feng Di , Krishnan Ramaswamy , Stella Lorenzo , Schenone Monica , Hartigan Chrisina R , Pollak Martin R.
Published: November 2023

α‐actinin‐4 gene (ACTN4) mutations cause a form of kidney injury called familial focal segmental glomerulosclerosis (FSGS) in humans. Individuals who carry disease‐causing ACTN4 mutations initially present with proteinuria due to podocyte injury, and many eventually develop end stage kidney disease. The disease‐causing ACTN4 mutations are exclusively located within the actin‐binding domain (ABD) of the encoded protein and result in increasing actin binding. We hypothesized that post‐translational modification of ACTN4 leads to similar biochemical and cellular changes observed in the disease‐causing ACTN4 mutant protein. Using mass spectrometry, we detected ACTN4 is phosphorylated at the serine 159 site (S159) in cultured human podocytes. By molecular dynamics simulations, we found that phosphorylated S159 causes a relative rotation of CH1 and CH2 domains within the ABD of ACTN4, potentially altering the actin binding interface. Supporting this is our observation that phosphomimetic S159D ACTN4 protein (which mimics the effect of phosphorylation at S159) increased F‐actin bundling activity in comparison with WT ACTN4, similar to the effect of kidney disease‐causing mutant K255E ACTN4 in vivo. Furthermore, when expressed in cultured human podocytes, phosphomimetic S159D ACTN4 led to similar changes as seen in the K255E mutant ACTN4: more prominent formation of actin stress fibers and significantly higher contractile force measured by traction force microscopy. We conclude that phosphorylation of ACTN4 at S159 induces characteristics similar to the kidney disease‐causing ACTN4 mutant both in vitro and in vivo. These data provide evidence that phosphorylation of ACTN4 plays an important role in regulating the cytoskeletal dynamics and function of podocytes.

Support or Funding Information

R37DK059588 and T32DK007199
Integrating GWAS And Experimental Data To Predict Targets And Models For Mechanistic Validation Of Genetic Determinants Of Salt Sensitive Hypertension
Authors: Hultström Michael , McBride Martin W.
Published: November 2023

The number of causal genes for human traits and diseases identified in GWAS increases steadily. However, only a small fraction has been successfully validated in experimental models to show how they may affect blood pressure regulation. Using recent hypertension GWAS we identified 1445 first and second tier SNPs with p‐values <10−5. Out of these 1379 could be annotated using SNP‐nexus and matched to 253 genes in Ingenuity Pathway Analysis (IPA).

The first experimental dataset used were the Mouse Diversity Array SNP data from the Mouse Genome Database for C57BL/6J that has a salt sensitive blood pressure, and Balb/c that is much less salt sensitive. This identified 6253 SNPs, whereof 4548 were annotated through SNP‐nexus. In IPA the SNPs could be matched to 406 genes. Three genes with different alleles between the mouse strains were also found in the 253 genes from the GWAS dataset (HHAT, PARK2, ABCC2).

Salt‐sensitive hypertension is usually thought to be associated to kidney function, and indeed all three gene‐products are detected by immunohistochemistry in renal tubuli in the proteinatlas.org database, where PARK2 also shows robust glomerular staining. Finally, in the results of an earlier micro array study we found that ABCC2 shows differential expression in the kidney between the strains, and is up regulated by a high‐sodium diet in C57BL/6J but not Balb/c indicating a functional difference.

ABCC2 deficient mice are available on the C57BL/6 background but do not seem to have any baseline differences in blood pressure or sodium balance. However, they have mostly been used to study bile acid, and have not been studied under high‐ or low‐sodium conditions.

In conclusion, ABCC2 is a second tier GWAS hit for hypertension in humans and appears amenable to mechanistic validation in the C57BL/6 mouse strain.

Support or Funding Information

Swedish Society of Medical Research
Role of BK in hypoxia‐induced elevation of [Ca2+]i in depolarized rat carotid body glomus cells
Authors: Wang Jiaju , Hogan James O. , Kim Donghee
Published: November 2023

The controversial issue on the role of BK in hypoxia‐induced depolarization of glomus cells is not fully resolved. Some studies show that BK is closed in resting isolated cells, whereas others show that BK is open and inhibited by hypoxia. Here we tested the possibility that BK may be active in a subpopulation of glomus cells because they are partially depolarized and/or the basal [Ca2+]i is high and that hypoxia further depolarizes these cells by inhibiting BK. Patch‐clamp single channel recording was used to assess BK activity and fura‐2 fluorescence intensity was recorded to assess [Ca2+]i. Depolarizing glomus cells with 20 mM [KCl]o did not activate BK in cell‐attached patches, indicating that depolarization up to ~40 mV was not sufficient to activate BK in intact cells. In cells perfused with 20 mM [KCl]o, hypoxia (solution bubbled with 0% O2 gas) activated BK, presumably due to the additional depolarization caused by hypoxia. In cells bathed in 5 mM KClo, blocking BK with TEA (3 mM) or iberiotoxin (100 nM) did not increase the basal [Ca2+]i. In cells bathed in 15 and 20 mM KClo, TEA did not alter the averaged [Ca2+]i, although some variable responses were observed among cells. Hypoxia produced a large increase in [Ca2+]i in all cells. These results show that in both undepolarized and partially depolarized glomus cells, BK makes little or no contribution to hypoxia‐induced excitation of glomus cells, but BK may help to limit the depolarization produced by hypoxia in partially depolarized cells.
Hot, Heat and Stretch All Contribute to the Enhanced Ca2+ Signaling in Pulmonary Arterial Hypertension
Authors: Song Shanshan , Yamamura Aya , Yamamura Hisao , Babicheva Aleksandra , Tang Haiyang , Ayon Ramon J , McDermott Kimberly M. , Sun Xutong , Black Stephen M. , Makino Ayako , Yuan Jason X.‐J.
Published: November 2023

Capsaicin is an active component of chili peppers and an irritant for mammals and humans. The dried chili pepper is a commonly consumed food ingredient and capsaicin is a potent inhibitor of substance P which is associated with inflammation and pain. Capsaicin is an important pain relief drug for patients with osteoarthritis pain, cancer‐associated pain and diabetic neuropathy‐mediated pain. In this study, we studied the effect of capsaicin on human pulmonary arterial smooth muscle cells (PASMC) from normal subjects and patients with idiopathic pulmonary arterial hypertension (IPAH). Our observations show that TRPV1, the capsaicin receptor and a transient receptor potential (TRP) cation channel in the TRP vanillioid family, is upregulated while capsaicin‐induced increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) is enhanced in IPAH‐PASMC compared with normal PASMC. TRPV1 is not only activated by capsaicin, but also activated by heat (>43°C or 102°F), acid (H+), and osmotic pressure changes. Indeed, the increasing temperature from 24°C to 43°C, or decreasing extracellular pH value from 7.4 to 5.0, both significantly enhanced capsaicin‐induced increases in [Ca2+]cyt. The heat‐(from 24°C to 43°C) and acidic pH (5.0)‐mediated enhancement of capsaicin‐induced increases in [Ca2+]cyt is greater in IPAH‐PASMC than in normal PASMC, potentially because of upregulated TRPV1 channels. These data indicate that upregulated TRPV1 expression and subsequent enhancement of capsaicin‐, heat‐ and acid‐mediated increases in [Ca2+]cyt in PASMC may play an important role in the development and progression of pulmonary arterial hypertension by inducing sustained pulmonary vasoconstriction and pulmonary vascular remodeling, while blockers of TRPV1 (e.g., capsazepine, yohimbine and cinnamides) can potentially be new therapies for PAH.
Calcium‐sensing Receptor, Coupled with Transient Receptor Potential Canonical Channels 6, Regulates Cytosolic [Ca2+] in the Development and Progression of Pulmonary Hypertension
Authors: TANG HAIYANG , Yamamura Aya , Fraidenburg Dustin R , Song Shanshan , Yamamura Hisao , Chen Jiwang , Gu Yali , Ayon Ramon J , Black Stephan M , Garcia Joe G.N. , Makino Ayako , Yuan Jason X.‐J.
Published: November 2023

Rationale

Elevated pulmonary arterial pressure (PAP) in patients with pulmonary arterial hypertension (PAH) is mainly caused by increased pulmonary vascular resistance (PVR), due primarily to sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling. An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMCs) is not only a major trigger for pulmonary vasoconstriction but also an important stimulus for pulmonary vascular remodeling. Our previous data showed that the extracellular calcium sensing receptor (CaSR) expression and function were both enhanced in PASMC from idiopathic PAH patients. However, the implication of CaSR in the development of PAH remains elusive.

Methods

Casr double knockout mice (Casr−/−Pth−/−) and transient receptor potential canonical Channels 6 (Trpc6−/−) have been exposed to hypoxia for 4 weeks to develop pulmonary hypertension. Following hypoxic exposure, right ventricle systolic pressures (RVSP), right ventricular hypertrophy by RV/(LV+S) ratios, and small pulmonary artery smooth muscle layer thickness were measured. The PAP was measured using the isolated perfused/ventilated mouse lung system. PASMCs were isolated from patients with IPAH, followed with [Ca2+]cyt measurement, proliferation and migration assays.

Results

In the current study, we found blockade of TRPC6 channels significantly inhibits Ca2+‐induced increase in [Ca2+]cyt and OAG caused a larger increase in [Ca2+]cyt in IPAH PASMCs. Overexpression of CaSR and TRPC6 channels increase the extracellular Ca2+‐induced [Ca2+]cyt in normal PASMCs. Compared to wild mice, both Casr−/−Pth−/−and Trpc6−/− mice had significantly lower RVSP, less right ventricular hypertrophy, and displayed less pulmonary vascular remodeling after exposure to hypoxia. Deletion of Casr or Trpc6 gene significantly attenuates acute hypoxic vasoconstriction.

Conclusions

These data indicate that CaSR functionally interacts with TRPC6 and regulate [Ca2+]cyt in IPAH‐PASMC. Deletions of CaSR or TRPC6 prevent the development of hypoxia‐induced PAH in mice via its modulation of extracellular Ca2+ entry in PASMC, suggesting that CaSR may be a novel therapeutic candidate for the treatment of PAH.
Hypoxia Decreases Trx2 Protein and Activity in Pulmonary Vascular Cells
Authors: Wade Brandy E , Adesina Sherry E. , Hart C. Michael , Sutliff Roy L.
Published: November 2023

Pulmonary Hypertension (PH) is characterized by a sustained increase in pulmonary arterial pressure. Increased pulmonary vascular pressure and resistance result in right ventricle (RV) hypertrophy and can ultimately lead to RV failure and death. The mechanisms leading to vascular cell proliferation and vascular remodeling in PH are incompletely defined. Current therapeutic approaches that stimulate vasodilation inadequately address the cellular proliferation and vascular remodeling that underlies the pathogenesis of PH. To more directly address mechanisms of cell proliferation in PH, this project focuses on the ubiquitin proteasome system (UPS) which plays an important role incellular homeostasis by regulating protein stability. Current evidence demonstrates changes in UPS activity in PH; however, the pathways and proteins influenced by these changes are poorly defined. Mice exposed to normoxic or hypoxic conditions for 3 weeks were used to identify proteins with altered ubiquitination in response to hypoxia exposure. Lungs were collected, and the PTMScan® Ubiquitin Remnant Motif (K‐ɛ‐GG) Kit was used to precipitate ubiquitinated proteins for analysis by mass spectrometry (MS). 245 proteins were identified with a ±2 fold change. In silico analysis identified several proteins that promote cell proliferation. Tax1bp1, Hspa8, Hspb1, Fhl1, Gnb1 interact with important cellular proliferation pathways and exhibit decreased ubiquitination in lungs from mice exposed to hypoxia, suggesting an increase in their stability in hypoxia. These results using MS and in silico analysis suggest that hypoxia‐induced changes in the UPS may impact proteins involved in pathways that regulate cellular proliferation by increasing or decreasing their stability.

Support or Funding Information

This work was supported by NIH RO1 HL 102167 and the T32 Training Program in Academic Pulmonary Medicine (T32HL076118).
Hypoxic pulmonary hypertension is not affected by neonatally induced diabetes in rats
Authors: Chalupsky Karel , Vajnerova Olga , Kratzerova Tereza , Durisova Jana , Mikova Dana , Rachacova Pavla , Sedlacek Radislav , Herget Jan , Hampl Vaclav
Published: November 2023

Chronic hypoxia elicits pulmonary hypertension, in part through oxidative alterations of the vascular wall. Since diabetes is also characterized by oxidative stress, we hypothesized that diabetes would amplify hypoxic pulmonary hypertension. We measured lung hemodymamic parameters, heart morphology and serum malonyldialdehyde (MDA, marker of oxidative burden) at the end of a 2‐week exposure of 11 weeks old diabetic rats to hypoxia (12% O2). Diabetes (glycemia >8 mM) was induced in the neonatal period by streptozotocine (100 mg/kg s.c.). Chronic hypoxia elevated pulmonary artery pressure (PAP) and right ventricle weight/left ventricle weight ratio (RV/LV+S), and this was not affected by diabetes. Diabetes alone increased PAP to a similar extent as chronic hypoxia, but this was solely due to elevated cardiac output. Serum MDA was elevated by diabetes and not significantly altered by hypoxia. We conclude that the oxidative insults of chronic hypoxia and diabetes to the pulmonary vascular bed are not additive.

Support or Funding Information

This material is based upon work supported by the project COST LD 14068, IGA NT 13358, GACR 13‐01710S and by the project „BIOCEV ‐ Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University” (CZ.1.05/1.1.00/02.0109) from the European Regional Development Fund.
The Geometry of the Progression of Occlusive Lesions in Severe Pulmonary Arterial Hypertension
Authors: Francis Christopher Michael , Oshima Kaori , Stevens Troy , Oka Masahiko
Published: November 2023

The vasculopathy of pulmonary arterial hypertension (PAH) includes the hallmark of occlusive, plexogenic lesions in pulmonary microvessels. Traditional studies of vascular lesions in PAH have relied on two‐dimensional histological preparations to grade and characterize lesion incidence and severity. However, this approach fails to acknowledge the physiological structure of vascular lesions in situ, and may systematically misidentify key anatomical features of the lung vasculature such as supernumerary arteries and vessel branching patterns. Therefore, we employed a three‐dimensional approach to composite serial histological lung sections to examine the geometry of vascular lesions in PAH. In light of recent three‐dimensional studies of vascular morphology in PAH which demonstrate that the progression of occlusive lesions has both temporal and spatial components, we tested the hypothesis that occlusive lesions within pulmonary microvessels in PAH exhibit patterns of severity along the length of vessel segments. Using Sugen/hypoxia treated hypertensive rats, we found that the incidence of occlusive lesions was undercounted with a conventional histological approach, due to the inability of cross‐sectional analysis to identify vascular trees linked in three‐dimensional space. Furthermore, we found that lesion severity was a complex function of vessel diameter, where disorganized lesions increased with decreased vessel diameter but were surrounded by a continuum of lesion grades. These results suggest that the contribution of occlusive lesions to the pathophysiology of PAH may be under represented, and underscore the necessity of three‐dimensional anatomical characterization of PAH vasculopathy.

Support or Funding Information

This work was funded by NIH grants HL60024 and HL66299
Ventilatory and Cerebrovascular Responses to Hypercapnia in Pulmonary Arterial Hypertension
Authors: Malenfant Simon , Brassard Patrice , Paquette Myriam , Le Blanc Olivier , Chouinard Audrey , Bonnet Sébastien , Provencher Steeve
Published: November 2023

BACKGROUND

Patients with pulmonary arterial hypertension (PAH) exhibit excessive ventilation at rest and during exercise. This is generally attributed to an increased physiological dead space related to extensive vascular remodeling and ventilation‐perfusion mismatch as well as hyperventilation resulting in low arterial carbon dioxide partial pressure (PCO2). However, mechanisms accounting for this hyperventilation remain elusive. We hypothesized that PAH patients exhibit enhanced central chemoreflex sensitivity, resulting in increased ventilator drive, hyperventilation and reduced cerebral blood flow.

METHODS

Central chemoreflex sensitivity was evaluated in 11 PAH patients and 11 healthy controls matched for age, sex and body mass index, using the modified hyperoxic hypercapnic rebreathing method. Participants underwent a 5‐minute period breathing room air. Then, they were asked to hyperventilate for one minute to an end‐tidal PCO2 (PETCO2) of 20–25 mmHg, before they started to breathe in a rebreathing bag (air mixture containing 7% CO2 and 93% O2) until PETCO2 reached 60 mmHg. During this protocol, left middle cerebral artery mean flow velocity (MCAvmean; transcranial Doppler), mean arterial pressure (MAP; finger photoplethysmography), PETCO2 and minute ventilation (VE,) were continuously monitored. The linear regression slope of VE‐PETCO2 characterized central chemoreflex sensitivity during the rebreathing period. Associations between central chemoreflex sensitivity, resting VE and MCAvmean were explored (Pearson's correlations).

RESULTS

Central chemoreflex sensitivity was significantly higher in PAH patients compared to controls (2.1 (0.3) vs 0.9 (0.2) L/min/mmHg; p=0.003). PAH patients also displayed lower PETCO2 (37 (5) vs 43 (5) mmHg; p=0.01) and increased VE (14 (2) vs 10 (2) L/min; p=0.0003) at baseline compared to controls. Central chemoreflex sensitivity correlated with resting VE (r=0.68; p=0.0005) and also inversely correlated with MCAvmean (r=−0.48; p=0.02), which was lower in PAH patients (59.3 (10.6) vs 74.2 (15.8) cm/s; p=0.01).

CONCLUSION

These results suggest that PAH patients exhibit reduced cerebral blood flow as a result of higher chemoreflex sensitivity and secondary hyperventilation. Alternatively, the reduction in cerebral blood flow induced by hypocapnia might decrease brain tissue carbon dioxide wash‐out, acidifying the cerebrospinal fluid and leading to a higher chemoreceptor stimulus and increased ventilation.

Support or Funding Information

Doctoral scholarship from Fond de Recherche du Québec ‐ Santé (FRQ‐S) to Simon Malenfant
eNOS Methylation at R494 is Increased With Hypoxia Exposure and May Be Associated with Decreased NO Production
Authors: Chicoine Louis G , Rafikov Ruslan , Black Stephen , Nelin Leif D , Cardounel Arturo J
Published: November 2023

Diminished endothelial nitric oxide (NO) biosynthesis is implicated in the pathogenesis of cardiopulmonary diseases. Much attention has focused on the production of endogenous eNOS inhibitors, such as ADMA, by protein‐arginine methyl transferases (PRMTs). Our recent studies demonstrate that in addition to generating free methylarginines, PRMTs can directly methylate arginine residues (R) in eNOS protein resulting in decreased NO production. In bovine pulmonary artery endothelial cell (bPAEC) studies siRNA was used to silence PRMT1 resulting in increased NO production and the finding that eNOS phosphorylation at serine 1177 was also increased compared to siRNA scramble treated cells. Furthermore, under hypoxic conditions we found that PRMT1 levels and activity were increased in bPAEC. We hypothesized that direct methylation of specific R residues in eNOS would be identified by mass spectroscopy (MS) and that the degree of eNOS methylation would increase with hypoxic exposure. Briefly, MS was applied to eNOS immune‐precipitated (IP) from bPAEC exposed for 48 hours to either normoxia (21%O2, 5%CO2) or hypoxia (1%O2, 5%CO2). Peptide calibration standards and matrix CHCA were obtained from Applied Biosystems (Carlsbad, CA). All spectra were taken on an ABSciex 5800 MALDI‐TOF‐TOF mass spectrometer in positive reflector mode (10 kV) with a matrix of CHCA. At least 1000 laser shots were averaged to obtain each spectrum. Masses were calibrated to known peptide standards. Five μl aliquots of the tryptic digest of the eNOS band (eNOS IP 18 h incubation with 10 ng MS grade trypsin at 28ºC in 25mM ammonium bicarbonate) were cleaned on a C18 ZipTip (Millipore, Bedford MA) as per manufacturer's instructions. Bound peptides were desalted with two 5μl washes of 0.1% TFA and then eluted with 2.5μl of aqueous, acidic acetonitrile (75% CH3CN, 0.1% TFA). The eluent was mixed with 2.5μl of freshly prepared CHCA stock solution (20 mg/ml CHCA in aqueous acetonitrile as above), and 1.5μl portions of this mixture were spotted onto a MALDI sample plate for air‐drying. 1.5μl of crude peptides were additionally mixed with 1.5μl of CHCA and spotted. MS and MS/MS spectra (1018.56 m/z corresponded to 484‐GSATKGAGITR*‐494 (*‐dimethylation site) sequence from eNOS) were analyzed in Protein Pilot 3.0 and Mascot Distiller software packages. We found that eNOS R494 is di‐methylated and that the amount of methylation increases (1.5 fold) with prior cellular exposure to hypoxia. Of note, this eNOS di‐methylated arginine is within the eNOS calmodulin‐binding region. Taken together, these results show that eNOS is indeed a substrate of PRMT1 and that posttranslational modification of R494 is likely to regulate eNOS activity.
Metabolic syndrome and heart failure with preserved ejection fraction: a new model of pulmonary hypertension associated with left heart disease
Authors: Potus Francois , Ranchoux Benoit , Tremblay Eve , Provencher Steeve , Bonnet Sébastien
Published: November 2023

Rationale

Heart failure (HF) with preserved ejection fraction (HFpEF) is the more common cause of HF and affecting over 50% of all patients with HF. Interestingly 50% of HFpEF culminates to pulmonary hypertension (PH), which confers poor prognosis. On the other hand, metabolic syndrome (MS), which affects more than 34% of adults in the world, is strongly associated with PH (pulmonary venous hypertension (94.1%); pulmonary arterial hypertension (34.3%)) and HFpEF (prevalence of 35%). Taken together, these results suggest that MS and HFpEF, which are two major healthcare challenges in the occidental society, may predispose to PH. Unfortunately; studies supporting this hypothesis remain limited by the lack of good pre‐clinical models that preclude extensive investigation. We developed a new animal model for WHO2 pulmonary hypertension associating HFpEF and metabolic syndrome.

Methods/Results

Using supra‐coronary aortic banding, we first induced HFpEF in wistar male rats characterized by left ventricular (LV) hypertrophy, raise in LV systolic pressure, LV end‐diastolic pressure and no significant modification of LV end‐diastolic area and LV ejection fraction, 5 weeks post‐surgery (n=5; p<0,05). We then induced MS with high fat diet and daily injection of olanzapine (4mg/kg for 21 days) initiated two weeks after surgery. Establishment of MS was confirmed by significant increase in the fat/lean body mass (minispec Brucker lf90) and insulin resistance (high blood insulin level) compared to vehicle‐treated rats (n=5 per group; P<0.05). As expected, compared to healthy and sham surgery, HFpEF rats (n=5) displayed PH characterized by a significant decreased pulmonary acceleration time and tricuspid annular plane excursion (PAAT; TAPSE; echocardiography; P <0.05) and increased mean pulmonary arterial pressure and right ventricular systolic pressure (PAP, RVSP; P<0.05) (right heart catheterizations in closed chest). Interestingly, MS‐HFpEF displayed significant worsening in hemodynamic parameters (PAAT, TAPSE, PAP, RVSP) compared to HFpEF rats without MS (P<0.05, n=5 per group). Interestingly, remodelling of the distal pulmonary arteries, strongly correlates with fat/lean body mass and was significantly increased in MS‐HFpEF rats compared to the other conditions (p<0.05, n=5 per group).

Conclusion

In the present study we demonstrated that metabolic syndrome worsens PH associated with HFpEF. Therefore, the MS and HFpEF combination represents a novel and attractive animal model to study WHO2 PH.
Aquaporin 1 (AQP1) Elevates cMyc and Cyclin D1 via β‐Catenin in Pulmonary Arterial Smooth Muscle Cells (PASMCs)
Authors: Yun Xin , Jiang Haiyang , Lai Ning , Shimoda Larissa
Published: November 2023

Pulmonary hypertension is a severe pulmonary vascular disease characterized by functional and structural changes in the vessels. During development of hypoxic pulmonary hypertension, primary features of remodeling in distal small pulmonary arteries include PASMC proliferation and migration. Formerly, we showed that hypoxia increased the expression of a water channel, AQP1, which mediates PASMC proliferation and migration by increasing β‐catenin protein levels. β‐catenin is a dual function protein that may interact with AQP1 in certain cell types and activates the transcription of crucial target genes related to cell migration and proliferation. Thus, we determined whether the AQP1 C‐terminal tail was required for AQP1 to increase β‐catenin levels in PASMCs and, if so, whether known β‐catenin target genes, such as c‐myc and cyclin D1, were also elevated. Primary cultured rat distal PASMCs were harvested and infected with adenoviral constructs expressing wild‐type AQP1 (AdWT), AQP1 lacking the C‐terminal tail (AdCT), or green fluorescent protein (AdGFP), as a control for infection. Compared to AdGFP cells, infection with AdWT, but not AdCT, increased β‐catenin protein and the mRNA and protein expression of c‐myc and cyclin D1. Moreover, knockdown of β‐catenin in PASMCs infected with AdWT prevented the AQP1‐induced increase in proliferation and migration.

Conclusions

Increased AQP1 enhances PASMC proliferation and migration through a mechanism involving modulation of β‐catenin protein levels and its targets via the C‐terminal tail of AQP1.
Sympathetic Nerve Activity and Baroreflex Dysfunction Paralleled the Progression of Pulmonary Arterial Hypertension in Monocrotaline Induced Rat Model
Authors: Shinoda Masako , Oga Yasuhiro , Saku Keita , Abe Kohtaro , Tohyama Takeshi , Kuwabara Yukimitsu , Yoshida Keimei , Takehara Takako , Fujii Kana , Nishizaki Akiko , Kishi Takuya , Ide Tomomi , Sunagawa Kenji
Published: November 2023

Background

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by progressive pulmonary vascular remodeling and extremely high pulmonary artery (PA) resistance. Although patients with advanced PAH are known to have excessive sympathoexcitation and baroreflex dysfunction, the relationship between autonomic dysfunction and the progression of PAH remains unknown. Thus, we investigated how the progression of PAH alters sympathetic nerve activity (SNA) and baroreflex function in monocrotaline (MCT) induced PAH rats.

Methods

In 7 weeks‐old Sprague‐Dawley rats (220–260g), we subcutaneously injected MCT (60 mg/kg) or saline for control (CTL). One, 3 and 3.5 weeks after MCT injection (MCT1W, MCT3W and MCT3.5W, respectively), we assessed right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH) (,‐the weight ratio of right to left ventricular) to evaluate the severity of PAH. We estimated SNA by 24 hours urine norepinephrine (U‐NE) and plasma norepinephrine (P‐NE). To measure open loop baroreflex function, we isolated bilateral carotid sinus regions and changed carotid sinus pressure (CSP) while measuring splanchnic SNA and arterial pressure (AP).

Results

RVSP significantly increased 3 and 3.5 week after MCT injection (CTL: 39±6, MCT3W: 73±6, MCT3.5W: 96±9 mmHg, p<0.05). RVH markedly increased in MCT3.5W (CTL: 0.29±0.04, MCT3.5W: 0.49±0.08, p<0.05). Both U‐NE and P‐NE paralleled RVSP (Fig. 1). In the baroreflex open loop analysis, increasing in CSP sigmoidally decreased SNA (neural arc), whereas SNA lineally increased AP (peripheral arc). The relationship between CSP and AP (i.e., baroreflex total loop function) was sigmoidal in both CTL and MCT3W, while the baroreflex induced maximum AP was lower in MCT3W than in CTL (Fig. 2). PAH lowered the baroreflex total loop gain at the operating point (CTL: −1.22±0.22, MCT3W:−0.35±0.14, p<0.05) (Fig. 3).

Conclusion

Sympathetic activation paralleled the progression of PAH. RVH and excessive SNA may deteriorate the baroreflex function in rats with advanced PAH.