bluuebunny/crossref_metadata_2025 huggingface dataset:
GENDER DIFFERENCES INVOLVING SEROTONIN (5‐HT) RECEPTORS IN TYPE I DIABETIC RATS
Authors: Moldovan Tudor , Cervantes Marisa , Bucan Jonathan , Walker Zachary , LePaige Bernard , Bhaskaran Subha , Banes‐Berceli Amy
Published: November 2023

Diabetes mellitus is an important topic of research. In both Type I and Type II diabetes vascular and renal dysfunction are some of the many negative impacts observed in diabetic patients. Increased plasma levels of 5‐HT exist in male diabetic rodent models and previous data in male Japanese diabetic patients demonstrated that inhibition of 5‐HT2A receptors reduced proteinuria observed. Whether these are differences in males and females in 5‐HT levels and function is unknown. We hypothesized that increased levels 5‐HT receptors may be the cause of the vascular damage observed in diabetics; elevated levels of 5‐HT and 5‐HT receptors may lead to increased vasoconstriction. We used male and female Sprague‐Dawley rats (300–325g) and made them diabetic with Streptozotocin (STZ). At 14 days and 28 days post‐onset of diabetes we euthanized the animals, harvested tissues and blood vessels for Western blot and myograph analysis. At day 14, no significant differences in contractile responses in the blood vessels from either the male or female control vs diabetic rats in the thoracic aorta, renal, superior mesenteric, and femoral arteries existed. There was a leftward shift in the response curve to 5‐HT in the vessels from the males of both groups compared to the females. At day 28, there was an increased contractile response to 5‐HT in the aorta from the diabetic rats compared to control from male and females. The females also showed increased contraction to 5‐HT in the superior mesenteric artery at 28 days. There was no change observed in the renal artery or femoral artery from either sex at 28 days. We observed at days 14 and 28 in the aorta of the male rats that there was no differences in 5‐HT2A or 5‐HT2B receptor levels. In the kidney cortex at day 14 we observed increased expression of both 5‐HT2A and 5‐HT2B receptor levels. At day 28 only the 5‐HT2B receptor in the kidney cortex from the diabetic rats was increased. There was no difference in the 5‐HT2A receptor levels. In the aorta from the 14 day female rats we saw greater expression of the 5‐HT2A receptor in the diabetic rats compared to control and less expression of the 5‐HT2B receptor in the aorta from the diabetic rats compared to control. At 28 days we observed no difference between groups in the 5‐HT2A receptor levels but a small increase in the levels of the 5‐HT2B receptor in the aortas from the diabetic rats. In the cortex from the female rats we saw increased levels of the 5‐HT2A receptor at both 14 and 28 days and increased levels of the 5‐HT2B receptor levels at 28 days. These data clearly show altered expression of 5‐HT receptor during the development of diabetes and clear sex differences as well.

Support or Funding Information

Oakland University Center for Biomedical Research
A Novel Role for AT1R: Ang‐(1‐7)/Mas Signal Transduction
Authors: Exner Eric C , Stodola Timothy J , Hoffmann Brian R , Greene Andrew S
Published: November 2023

Microvascular dysfunction is a key component of cardiovascular disease associated with many risk factors, including hypertension and diabetes. In models of renin suppression and hypertension, we have observed that microvascular function can be rescued by infusion of low doses of angiotensin‐(1‐7) (Ang‐(1‐7)). In previous studies, we detected the angiotensin II receptor type 1 (AT1R) as a potential interacting mechanism for transduction of signals caused by the binding of Ang‐(1‐7) to the Mas receptor. While the oligomerization of AT1R and other G‐protein coupled receptors has been described, the various partner receptors and physiological roles are just beginning to be illuminated. Here, we investigate whether AT1R is necessary for the effects of Mas mediated Ang‐(1‐7) signaling on microvascular function in a model of renin suppression and salt‐sensitive hypertension. We also seek to identify downstream mediators of Ang‐(1‐7)/Mas signaling that are disrupted in the absence of AT1R. In an AT1R knockout rat, the ability of Ang‐(1‐7) to rescue microvascular function in a model of hind‐limb angiogenesis was lost. In addition, Ang‐(1‐7) mediated enhancement of endothelial cell tube formation was compromised in AT1R knockout cells in vitro. AT1R dependent downstream mediators of Ang‐(1‐7)/Mas signaling were identified by combining high throughput, mass spectrometry based proteomics with gene expression data. These findings, combined with our previous work showing the presence of AT1R in the Ang‐(1‐7)/Mas signaling complex, suggest that physical proximity of Mas and AT1R is important for signal transduction. Further studies will validate targets identified here and investigate the role of Ang‐(1‐7) and the Mas/AT1R complex in regulation of endothelial progenitor cell function. These results suggest that oligomerization of receptors within the RAS may be of clinical importance, as regulation of microvascular function via the RAS is both pathophysiologically and pharmacologically relevant.

Support or Funding Information

This work was supported by NHLBI grants HL082798, T32HL7852‐18 and F30HL131153 as well as the Kern Foundation Innovation Award granted to Dr. Andrew S. Greene.
Zinc‐finger nuclease knockout of dual‐specificity protein phosphatase‐5 protects against DOCA‐salt induced chronic renal injury
Authors: Fan Fan , Murphy Sydney R , Ge Ying , Mims Paige N , Maeda Kenji J , Williams Jan M , Roman Richard J
Published: November 2023

We recently reported that zinc‐finger nuclease knockout (KO) of dual‐specificity protein phosphatase‐5 (Dusp5) enhances the myogenic response and autoregulation of cerebral blood flow in FHH.1BN rats. We identified 4 single nucleotide polymorphisms in Dusp5 in FHH as compared with BN rats, two of which alter CpG sites and another one causes G155R mutation. In the present study, we evaluated whether Dusp5 also plays a role in the regulation of autoregulation of renal blood flow and renal function in a Dusp5 knockout (KO) rat in the FHH.1BN genetic background, which an 11 bp frame‐shift deletion is introduced by zinc‐finger nuclease and a premature stop codon at AA 121 of Dusp5 protein is produced. The expression of Dusp5 was lower in FHH and in Dusp5 KO rats in comparison to the FHH.1BN rats. The level of phosphorylated ERK1/2 (p‐ERK1/2), a substrate of Dusp5, was enhanced in the KO rats. The blood pressure and urinary protein excretion was similar in Dusp5 KO (n = 43) and FHH.1BN rats (n = 30) when they were at 12‐week of age (114 ± 2 vs. 115 ± 1 mmHg and 32 ± 2 vs. 29 ± 3 mg/day). In response to an elevation in pressure from 60 to 120 mm Hg, the renal afferent arterioles (Af‐arts) constricted by 29 ± 2% and 11 ± 1%, and the vascular tone was increased to 36 ± 2% vs 19 ± 1%, respectively in Dusp5 KO (n = 6) in comparison to FHH.1BN rats (n = 17). Blood pressure was 169 ± 5 vs. 177 ± 5 mmHg in Dusp5 KO (n = 11) vs. FHH.1BN rats (n = 14) after 3‐week DOCA‐salt treatment. However, proteinuria was significantly reduced in Dusp5 KO rats (258 ± 22 mg/day, n = 12) in comparison to FHH.1BN rats (338 ± 30 mg/day, n = 12) with the same treatment. The renal injury induced by DOCA‐salt was markedly reduced in Dusp5 KO vs. FHH.1BN rats as the glomerular injury scores were 2.49 ± 0.03 vs. 2.54 ± 0.01; the areas of renal fibrosis were 5.5 ± 0.5% vs. 8.3 ± 0.3%; the areas of renal protein casts were 0.5 ± 0.2% vs. 2.4 ± 1.0% and renal arteriolar media‐to‐lumen ratios were 0.64 ± 0.03 vs. 0.81 ± 0.04, respectively. These results indicate that Dusp5 modulates myogenic reactivity in the renal circulation and protects against DOCA‐salt hypertension induced chronic renal injury. It supports the view that upregulation of the expression of Dusp5 and/or an activating mutation in Dusp5 may contribute to the impaired myogenic response and autoregulation of renal blood flow and promote the development of chronic renal injury in FHH rats.

Support or Funding Information

This study was supported by grants HL36279 (RJR) and DK104184 (RJR), AG050049 (FF), P20GM104357 (RJR, FF and JMW) from the National Institutes of Health; 16GRNT31200036 (FF) and 12SDG9440034 (JMW) from the American Heart Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
mTORC1 Signaling Is Required for Leucine to Induce Endothelial Dysfunction in the Mouse Aorta
Authors: Reho John J , Guo Deng‐Fu , Rahmouni Kamal
Published: November 2023

Obesity‐induced hypertension is associated with vascular endothelial dysfunction. Our laboratory has previously demonstrated a critical role of the mammalian target of rapamycin complex 1 (mTORC1) in the regulation of the cardiovascular system and arterial pressure. mTORC1 serves as an intracellular energy sensor regulating protein synthesis through its downstream signaling components S6‐kinase and the ribosomal S6 protein. We tested the hypothesis that activation of the mTORC1 signaling pathway in the vasculature is involved in the regulation of vascular endothelial function. Aortic rings isolated from wildtype male mice were stimulated with the branched chained amino acid leucine ex vivo (10mM; 24hrs) to activate mTORC1 signaling. Leucine activated mTORC1 signaling in the aorta as indicated by the increased phosphorylation of the downstream S6 via Western blot (2.0±0.2AU vs 1.0±0.2AU in unstimulated controls; p<0.05). Immunohistochemistry revealed that mTORC1 signaling was increased in both the endothelium and the smooth muscle of aortas exposed to leucine stimulation. Leucine stimulated aortic rings exhibited impaired acetylcholine induced relaxation compared to control (Max. relaxation: 71±4% vs 56±3%; p<0.05) with no changes in relaxation responses evoked by sodium nitroprusside (Max. relaxation: 84±2% vs 78±4%) indicating endothelial but not smooth muscle dysfunction. We next infected leucine stimulated aortic rings with an adenoviral S6‐kinase dominant negative construct (Ad‐DNS6K; 24hrs) and this rescued the endothelial dysfunction associated with leucine induced mTORC1 activation in aortic rings (Max. relaxation: 69±2%; p<0.05) with no effect on relaxation responses to sodium nitroprusside (Max. relaxation: 81±3%). To test if direct activation of mTORC1 signaling was involved in vascular endothelial function, we used an adenoviral construct of a constitutively active S6‐kinase (Ad‐CAS6K) to recapitulate leucine‐induced endothelial dysfunction in aortic rings. Infection of aortic rings with Ad‐CAS6K increased the phosphorylated S6 protein compared to adenoviral GFP (Ad‐GFP) control (2.1±0.1AU vs 1.0±0.1AU; p<0.05). This activation of mTORC1 signaling resulted in impaired acetylcholine‐induced relaxation (Max. relaxation: 67±5 vs. 81±3%; p<0.05) without altering the relaxation evoked by sodium nitroprusside (Max. relaxation: 90±1% vs. 90±2%) recapitulating the vascular phenotype in leucine stimulated aortic rings and obese mice. We conclude that mTORC1 signaling is a novel regulator of vascular endothelial function and that dysregulation of this signaling pathway may be involved in the endothelial dysfunction associated with states of overnutrition such as obesity.
ERalpha Protects Against Lipoprotein‐Associated Oxidative Stress to Regulate Vascular Tone in Both Male and Female Mice
Authors: Kim Seong Chul , Lee Jean‐Pyo , Hamblin Milton H.
Published: November 2023

Lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1) is a major vascular scavenger receptor for binding and cellular uptake of oxidized low‐density lipoproteins (oxLDL). LOX‐1 is an important mediator of oxLDL‐stimulated pathways that trigger vascular dysfunction, inflammation, and oxidative stress. However, the mechanisms protecting against lipoprotein‐related oxidative stress to maintain normal vascular tone are understudied. Here we investigated the connection of ERalpha‐activated vascular protection with the oxidative status of vascular rings. We preincubated male and female aortic vessels with 10 μM ox‐LDL for 30 minutes, and after measuring phenylephrine contractile responses, treated the aortas with 10−12 to 10−5 μM of 4,4′,4″‐(4‐propyl‐[1H]pyrazole‐1,3,5‐triyl)tris‐phenol (PPT), a specific ERalpha agonist. We found that PPT induced relaxation in aortic rings preincubated with ox‐LDL in both sexes, demonstrating a vascular protective role of ERalpha in lipoprotein‐associated oxidative stress. Given the interplay between ERalpha signaling and lipoprotein‐associated oxidative stress, we evaluated the expression level of LOX‐1 in PPT‐stimulated mouse endothelial cells in vitro. Western blot analysis shows that LOX‐1 expression was attenuated in endothelial cells following treatment with PPT. Collectively, these data suggest a protective role of ERalpha against lipoprotein‐associated oxidative stress in regulating vascular tone. Moreover, we anticipate these studies will provide a broader look into intracellular signaling mechanisms that influence vascular tone and events.

Support or Funding Information

Supported by 5K12HD043451
Cardiorespiratory Dysfunction in Advanced Lung Disease versus Systemic Lupus
Authors: Wooten Liana C , Chin Lisa M.K. , Collins John , Chan Leighton , Keyser Randall E.
Published: November 2023

The cardiorespiratory responses to a peak exercise challenge were compared among women manifesting three autoimmune mediated conditions, pulmonary hypertension (PH), interstitial lung disease (ILD) and systemic lupus erythematous (SLE), and sedentary but otherwise healthy controls.

Methods

80 women (PH N=28, age=54.0 ± 10.3, BMI=31.8 ± 7.3; ILD N=9, age=55.8 ± 7.2, BMI=29.7 ± 4.6; SLE N=25, age=35.1 ± 9.0, BMI=27.6 ± 5.8; Control N=18, age=36.8 ± 7.3, BMI=39.3 ± 8.8) completed a treadmill cardiopulmonary exercise test (CPET) to volitional exhaustion. Heart rate was obtained by continuous EKG monitoring and pulmonary gas exchange information was obtained using a breath‐by‐breath cardiopulmonary exercise testing system. Peak exercise oxygen consumption (VO2), heart rate (HR) and respiratory exchange ratio (RER) were recorded as an average of the last 8 consecutive breaths. Oxygen pulse (O2‐pulse) was calculated and the V‐slope method was used to determine VO2 observed at the anaerobic threshold (AT‐VO2). Data were analyzed for statistical significance (p<0.05) using analysis of variance and Cohen's d‐statistic for effect size.

Results

Peak RER was ≥ 1.10 in all groups except PH (RER = 1.00 ± 0.25). Peak VO2 and AT‐VO2 was significantly lower in the PH, ILD, and SLE groups compared to controls (p<0.01).

Peak VO2(ml/kg/min)
AT‐VO2(ml/kg/min)
Peak HR(beats/min)
O2‐Pulse(ml/beat)

PH
15.3 ± 5.4
10.3 ± 4.3
139 ± 17
9.2 ± 4.6

ILD
18.2 ± 5.9
11.7 ± 3.8
150 ± 19
9.6 ± 4.6

SLE
19.4 ± 4.8
12.5 ± 3.2
171 ± 18
8.3 ± 1.9

Control
28.8 ± 5.2
16.4 ± 2.2
180 ± 10
11.0 ± 2.3

Peak VO2 was lower in PH than in SLE (p=0.031), whereas AT‐VO2 was similar among PH, ILD and SLE. No difference in peak HR was observed between SLE and controls or between PH and ILD, however peak HR was significantly higher in SLE than in ILD (p=0.009) and PH (p=0.001). While not statistically significant, the size of the group difference in O2‐pulse was large (Cohen's d = 1.283).

Discussion

Previous studies have demonstrated similarity in peak cardiac output and stroke volume between healthy controls and patient subsets with autoimmune conditions such as PH, ILD and SLE. The decline in peak VO2 and HR across the PH and ILD groups, compared to controls, in concomitance with the disappearance of this decline with respect to O2‐pulse may suggest peripheral muscle fatigue as a factor limiting exercise tolerance in these groups. The large effect in SLE with respect to the decline in O2‐pulse and similarity in peak HR with controls may provide further evidence for peripheral oxygen extraction impairment in women with SLE.

Support or Funding Information

Supported by NIH/NICHD/NCMRR HD39775; NIH IRP 1 Z01 CL060068‐02 CC
Dysregulation of Vasodilatory Pathways is a Contributory Factor to Acute Chest Syndrome
Authors: Frazier Krystle , Young Sheila M , Cook Kayleigh , Ibe Basil Obijiaku
Published: November 2023

Acute chest syndrome (ACS), the second most common cause of hospitalization for sickle cell disease (SCD) patients and cause for 25% of premature death cases, remains a therapeutic challenge due to the unknown nature of the syndrome's development. SCD patients exhibit a baseline leukocytosis, or pro‐inflammatory state, that increases their risk of developing ACS. Red blood cells (RBC) of SCD patients induce injury to the lung microvasculature resulting in the release of clotting factors and inflammatory mediators. Our hypothesis is that these mediators sustain pulmonary endothelial injury and RBC adhesion to the vessel wall resulting in pulmonary vascular occlusion and ultimately ACS.

Objective

To investigate a mechanism by which RBCs stimulate production and release of inflammatory cytokines and proteins in SCD.

Methods

Studies were conducted on human pulmonary microvascular endothelial cells (HPMEC) with and without overlay with RBCs or neutrophils of SCD patients in the steady state. Inflammatory mediators were measured by ELISA or Western blotting. Data were analyzed statistically at p<0.05.

Results

HPMEC cultured with RBCs from SCD patients released 40% more leukotriene C4 and 6‐fold more 11β‐PGF2α and more TxA2 than controls. Release of prostacyclin by SCD RBC blood was not different from control.

Conclusions

Sickle cell mutation‐bearing erythrocytes cause release of inflammatory mediators that activate vasoconstrictive pathways in HPMEC and down‐regulate vasodilatory pathways. This condition may eventually overwhelm the ability of the HPMEC to secrete vasodilators. A closer look at the interaction of these mediators and microvascular endothelium may reveal genetic polymorphisms and provide a potential for therapeutic targeting with gene silencing technology.

Support or Funding Information

AMA Foundation
TRPV4 and TRPC1 Channels Mediate Hemodynamic Stress‐Induced Exacerbation of Pulmonary Hypertension
Authors: Sham James S.K. , Eldridge Lindsey , Paudel Omkar , Moldobaeva Aigul , Wagner Elizabeth
Published: November 2023

Pulmonary hypertension (PH) is a pathological condition associated with a wide spectrum of diseases with diverse etiologies. It is characterized by progressive pulmonary vascular remodeling, sustained vasoconstriction and enhanced vasoreactivity, resulting in elevated pulmonary vascular resistance and right heart failure. Recent studies suggest that increased hemodynamic stress/stretch in pulmonary vasculature plays important roles in vascular remodeling during the development of PH. However, the mechano‐transduction mechanism for hemodynamic stress in PH is unclear. Previous studies showed that several transient receptor potential (TRP) channels are mechanosensitive in different cell types. In particular, TRPV4, an osmo/mechanosensitive channel, is highly expressed in pulmonary endothelial and arterial smooth muscle cells (PASMCs). We have previously demonstrated that TRPV4 expression is upregulated in PASMCs during PH, and deletion of trpv4 gene delayed and suppressed PH development. Moreover, TRPV4 may form heterotetrameric channels with the store‐operated TRPC1, which is known to play crucial roles in PASMC proliferation and vascular remodeling. In this study, we sought to test the hypothesis that TRPV4 and TRPC1 are important components for mediating the signal of hemodynamic stress in vascular cells during PH. To test this hypothesis, left pulmonary arteries ligation (LPAL) was performed in WT, trpv4−/− and trpc1−/− mice to augment hemodynamic stress in pulmonary vasculature. After one week of recovery, LPAL and sham‐operated mice were exposed to normoxia, chronic hypoxia (CH, 3 week 10% O2), or treated with weekly injections of Sugen‐5416 (20 mg/kg) and exposed to CH for 4 weeks. LPAL had no significant effect on the right ventricular systolic pressure (RVSP) and right ventricular mass index (RVMI, RV/(LV+S)) in normoxic mice, suggesting that the increase in pulmonary blood flow in the right lung is well compensated under control conditions. By contrast, the increase in RVSP and RVMI induced by CH exposure or sugen/hypoxia treatment were significantly enhanced in the LPAL‐operated WT mice compared to the sham‐control, indicating that the increased hemodynamic stress exacerbates PH. More importantly, the PH and RV hypertrophy caused by CH were significantly attenuated in the sham‐operated trpv4−/− and trpc1−/− mice, and LPAL in both types of knockout mice did not caused further increase in RVSP and RVMI. These results, hence, support our hypothesis that TRPV4 and TRPC1 channels play an essential role in mediating the hemodynamic stress‐induced alterations in vascular properties during PH; and they may be considered as novel targets for comprehensive treatment of PH.

Support or Funding Information

This work is supported in part by NIH grant HL075134
Three‐dimensional Characterization of Occlusive Lesions in Pulmonary Arterial Hypertension
Authors: Francis Christopher Michael , Oshima Kaori , Baghaei Neelufar , Jeon Dayoung , Labat Sydney , Oka Masahiko , Stevens Troy
Published: November 2023

Pulmonary arterial hypertension (PAH) includes a vasculopathy of fixed occlusive lesions in small pulmonary arteries. Evaluation of vascular lesions in PAH has relied on two‐dimensional histological preparations and morphological grading methodologies to evaluate lesion incidence and severity. However, this approach fails to acknowledge the structure of vascular lesions in situ and is inherently prone to investigator bias, leading to controversy regarding the hemodynamic impact of occlusive lesions in PAH and the possible misidentification of lesions within the lung vasculature. Therefore, we developed a semi‐automated, three‐dimensional approach to the analysis of composite serial histological sections to examine the geometry of vascular lesions in PAH. Given that recent studies of vascular morphology in PAH demonstrate that the progression of occlusive lesions has both temporal and spatial components, we tested the hypothesis that occlusive lesions within pulmonary arteries in PAH vary in composition and severity along the length of vessel segments. Using Sugen/hypoxia treated PAH rats, we found that lesion composition and severity was a complex function of vessel length, where von Willebrand factor‐positive endothelium and luminal occlusion surrounding plexiform lesion sites formed a highly variable continuum. These results suggest that the contribution of occlusive lesions to the pathophysiology of PAH may be misrepresented, and underscore the necessity of an unbiased approach to the anatomical characterization of the vasculopathy of PAH.

Support or Funding Information

Supported by NIH HL66299 and HL60024.
Pulmonary artery input impedance reflects histological changes in the pulmonary artery in Sugen/Hypoxia pulmonary hypertension model
Authors: Nishikawa Takuya , Saku Keita , Kishi Takuya , Tsutsui Hiroyuki , Sunagawa Kenji
Published: November 2023

Background

Pulmonary hypertension (PH) is characterized by elevated pulmonary arterial pressure resulting from pulmonary vasoconstriction and remodeling, and leads to end‐stage right heart failure. Although pulmonary arterial resistance (R) has been a standard measure to characterize the mechanical properties of pulmonary arterial system, arterial input impedance (Z) provides far more detailed dynamic as well as static mechanical characteristics of pulmonary artery. We hypothesized that Z reflects histological changes in the pulmonary artery in PH.

Method

In Sprague‐Dawley rats, we induced PH by an injection of SU5416 (20 mg/kg) and 3 weeks exposure to 10% oxygen. We conducted acute experiments under anesthesia at 0, 1, 3 and 8 (n=7 each) weeks after SU5416 injection. We measured pulmonary artery flow (F) and pressure (P) under irregular pacing. We Fourier transformed both F and P to derive Z and fitted the 2‐element Windkessel model to Z to estimated R and elastance (E). After the hemodynamic measurements, we harvested the lung. In histology, we measured the pulmonary arterial cross sectional area within the external elastic lamina (AEEL) and that within the internal elastic lamina (AIEL) and indexed the loss of luminal area by LLA=(AELL‐AILL)/AELL.

Result

PH progressively increased mean P (Baseline: 18.8 ± 1.0, 1W: 26.0 ± 3.1, 3W: 45.6 ± 5.4, 8W: 63.9 ± 13.4 mmHg, p<0.01), whereas F remained unchanged (Baseline: 34.7 ± 5.9, 1W: 36.9 ± 7.4, 3W: 32.5 ± 4.5, 8W: 31.8 ± 8.7 ml/min, n.s.). This is translated into the progressive increases in Z without changing its dynamic properties (Fig. 1). Estimated R (Baseline: 17.4±3.9, 1W: 29.7±8.6, 3W: 55.7±9.0, 8W: 70.2±18.2 mmHg/ml/sec, p<0.01) and E (Baseline: 267±48, 1W: 424±101, 3W: 619±213, 8W: 709±239 mmHg/ml, p<0.01) (Fig. 2) increased, while the resultant time constant (R/E) remained unaltered. LLA of arteries with the outer diameter (OD) of <50μm (LLA50) increased with the progression of PH and reached more than 50% (Baseline: 0.18±0.01, 1W: 0.29±0.02, 3W: 0.43±0.06, 8W: 0.52±0.03, p<0.01). In contrast, LLA of artery with 50<OD<100 μm (LLA100) also increased with the progression of PH, but remained less than 30% (Baseline: 0.12±0.02, 1W: 0.20±0.03, 3W: 0.24±0.04, 8W: 0.27±0.03, p<0.01) (Fig. 2).

Conclusion

PH progressively increases R, E, LLA50 and LLA100 indicating arterial wall thickening and loss of luminal area. Taken together, in PH, we conjecture that increases in LLA50 contribute to increase R, whereas those in LLA100 may not contribute much to increase R. Increases in LLA100 at least in part contribute to increase E. Thus, Z closely couples with pulmonary arterial histology and may serve as a new tool to assess the disease progression in PH patients.
PHD2 Deficiency Induces Obliterative Vascular Remodeling and Severe PAH through Nitrative Stress and Caveolin‐1 Downregulation
Authors: Dai Zhiyu , Zhu Maggie M. , Zhao You‐Yang
Published: November 2023

Background

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by increased pulmonary vascular resistance and progressive vascular remodeling. Our recent study for the first time demonstrated that loss of hypoxia inducible factor (HIF) prolyl‐4 hydroxylase 2 (PHD2) in endothelial cells and bone marrow cells in mice induces severe PAH with obliterative pulmonary vascular remodeling and plexiform‐like lesions recapitulating clinical PAH. Increased oxidative/nitrative stress is a hall marker of clinical PAH and Caveolin‐1 (Cav1) is an important regulator of oxidative/nitrative stress and the pathogenesis of clinical PAH. It is unclear whether Cav1 deficiency and nitrative stress are involved in the pathogenesis of severe PAH in this mouse model.

Methods

Mice with Tie2Cre‐mediated disruption of Egln1 (encoding PHD2) (Egln1 CKO), Egln1 and Hif2a (EH2 DKO), and mice with Egln1 disruption and Cav1 transgene (expressing Cav1 in endothelial cells) (Egln1 CKO/Tg) were generated. Western blotting and immunostaining were performed to quantify nitration stress and Cav1 expression. MnTMPyP (a superoxide scavenger) and Tempol (a radical scavenger) were administrated to Egln1 CKO mice. Right ventricular systolic pressure (RVSP) and RV/(LV+S) ratio, indicator of RV hypertrophy, were measured. Pulmonary vascular remodeling were assessed by histological examination. Echocardiography was used to measure RV and PA function.

Results

Nitrotyrosine was markedly increased in Egln1 CKO mouse lungs and normalized in EH2 DKO mouse lungs. Both MnTMPyP and Tempol treatment inhibited RVSP increase and RV hypertrophy, and decreased medial thickness and muscularization of distal pulmonary arterials in Egln1 CKO mice. Mechanistically, endothelial Cav1 expression was decreased in Egln1 CKO mouse lungs and normalized in EH2 DKO mice. Restored Cav1 expression in Egln1 CKO/Tg mice inhibited RVSP and RV hypertrophy and resulted in normalization of RV and PA function and pulmonary vascular remodeling. Both pharmacological and genetic approaches promoted survival of Egln1 CKO mice.

Conclusion

These data show that loss of endothelial PHD2 suppresses Cav1 expression in a HIF‐2α dependent manner. Decrease of Cav1 expression and resultant activation of nitrative stress contribute to the severe vascular remodeling and PAH in Egln1 CKO mice. Thus, targeting nitrative stress and restoring Cav1 expression are potentially effective approaches for treatment of PAH.

Support or Funding Information

NIH grants R01HL123957, R01HL125350, R01HL133951, and P01HL077806 (Project 3) to Y.Y.Z, AHA postdoctoral fellowship grant 15POST25700124 to Z.D.
Effects of inhibition of MAPK14 and PDE4 signaling pathways on β‐aminopropionitrile fumarate‐induced thoracic aortic aneurysm and dissection in rats
Authors: WANG JIUN‐JR , Yang Yu‐Chun , Wu Nan‐Chun
Published: November 2023

Objectives

Thoracic aortic aneurysm (TAA) is a virulent and clinical silent disease. The overall mortality rate of a rupture or dissection of TAA could be above 90% in 30 days. TAA grows slowly, and 95% of people have no symptoms prior to its rupture or dissection. To date, treatment strategy for TAA has been limited by elective surgery, being put on observation with treatments of b‐blocking agents or endovascular stent‐graft therapy, while any surgery carries risks and the protective efficacy of b‐blockers or endovascular therapy is inconclusive. β‐aminopropionitrile fumarate (BAPN) is a potent irreversible inhibitor of lysyl oxidase, an enzyme initiating the crosslink formations between collagens and elastin, and mediating extracellular matrix remodeling. Using BAPN, we developed a rat model of TAA that leads to consistent aortic dissections. Genechip analysis of TAA tissues revealed highly upregulated mitogen‐activated protein kinases (MAPKs) and phosphodiesterases (PDEs) expressions. In this study, we aimed to examine efficacy of MAPK and/or PDE inhibition against the progression of TAA and its fateful consequence, dissections.

Methods

This study was conducted in 4 week old Sprague‐Dawley male rats, divided in two groups, sham and BAPN‐treated. The latter was further divided into 4 subgroups; they were treated intraperitoneally with p38 MAPK inhibitor (SB203580, 2 mg/kg, i.p.), PDE4D2 inhibitor (ibudilast, 5 mg/kg), SB203580+ ibudilast and equal amount of saline, respectively. We measured the body weight, food and water consumption daily, noninvasive tail‐cuff pressure every other day, and invasive ventricular pressure‐volume and aortic pressure and flow at 4 weeks. Aortic tissues were subjected to histological, transmission electron microscopic, protein and gene expression analysis.

Results

There were no major differences in body weight, food and water consumption among different groups. BAPN did not appear to affect ventricular function, but increased reflected waves and reduced aortic compliance. Fig. 1 showed that smooth muscle cells disintegrated and elastin and collagen fragmented en masse after 4 weeks' BAPN treatment. Either p38 MAPK or PDE4D2 inhibitor treatment attenuated structural deterioration, whereas treatment with both inhibitors exhibited synergetic protective advantages. MMP‐2, MMP‐9, COX‐2 and TGF‐β1 protein expression and NF‐κB and CAMK II gene expression were reduced by SB203580 or ibudilast, and most significantly by combined treatment.

Conclusion

MAPKs and PDEs may play an important role in the progression of vascular degeneration of TAA. Inhibitions of both p38 MAPK and PDE4D2 attenuated MMP‐2, MMP‐9, COX‐2 and TGF‐β1 protein and NF‐κB and CAMK II gene expressions and protected vascular structures.

Support or Funding Information

Supported by the Ministry of Science and Technology of Taiwan to Dr. Jiun‐Jr Wang (105‐2320‐B‐030 ‐008)
Involvement of inhibitor kappa B kinase 2 (IKK2) in the regulation of vascular tone
Authors: Youngin Kwon , Soo‐Kyoung Choi , Seonhee Byeon , Young‐Ho Lee
Published: November 2023

Inhibitor kappa B kinase 2 (IKK2) plays an essential role in the activation of nuclear factor kappa B (NF‐kB). Recently, it has been suggested that IKK2 acts as a myosin light chain kinase (MLCK) and contributes to vasoconstriction in mouse aorta. However, the underlying mechanisms are still unknown. Therefore, we investigated whether IKK2 acts as a MLCK or regulates the activity of myosin light chain phosphatase (MLCP). We used 12‐week old Sprague Dawley rats and measured vessel diameter of mesenteric artery using pressure myograph. SC‐514 (IKK2 inhibitor, 50 μM) induced relaxation in the mesenteric arteries pre‐contracted with 70 mM high K+ solution or U‐46619 (thromboxane analogue, 5 μM). The relaxation induced by SC‐514 was increased in the arteries pre‐contracted with U‐46619 compared to those with 70 mM high K+ solution. In the absence of intracellular Ca2+, U‐46619 still induced contraction, which was decreased by SC‐514. Furthermore, U‐46619‐induced contraction was decreased by SC‐514 in the presence of MLCK inhibitor ML‐7 (10 μM). We demonstrated that IKK2 is involved in the vascular contraction and contributes to regulation of MLCP.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science, ICT & Future Planning) (No. 2016R1C1B1006504).
The First Evidence of Decreased Voltage‐Gated Ca2+ Influx into Coronary Smooth Muscle in Ischemic Versus Non‐Ischemic Cardiomyopathy in Human Transplant Cardiectomy Samples
Authors: Badin Jill K. , Alloosh Mouhamad , Chen Robert J. , Hashmi Zubair A. , Sturek Michael
Published: November 2023

Ca2+ regulation is pivotal to coronary smooth muscle (CSM) cells. Most Ca2+ influx is routed via voltage‐gated Ca2+ channels (VGCC). A fraction of the intracellular Ca2+ is sequestered in the sarcoplasmic reticulum (SR) by the sarco‐endoplasmic reticulum Ca2+ ATPase (SERCA) for later release via ryanodine receptors (RyR). Evidence in animal models of atherosclerosis clearly shows that intracellular Ca2+ handling is dysregulated in metabolic syndrome. We tested the hypothesis that intracellular Ca2+ handling would be dysregulated more in CSM in human ischemic (atherosclerotic) vs. non‐ischemic cardiomyopathy. Freshly isolated CSM cells from human hearts excised in transplant cardiectomy were loaded with the fluorescent intracellular Ca2+ indicator fura‐2 and placed in a constant‐flow superfusion chamber. Membrane depolarization with 80 mM K+ induced Ca2+ influx largely via VGCCs and caused loading of the SR store. Caffeine‐induced SR Ca2+ store release and undershoot after store depletion (SERCA activity) were also measured. Pure VGCC activity was calculated as the slope of Ba2+ influx during superfusion with a 2 mM Ba2+ solution, as Ba2+ permeates VGCC but is not buffered by SERCA or Na‐Ca exchange. CSM isolated from hearts from patients with ischemic cardiomyopathy exhibited lower depolarization‐induced Ca2+ influx, similar SR store release, similar undershoot, and trended towards lower barium slope than CSM from non‐ischemic patients. Overall, Ca2+ influx via VGCC, but not Ca2+ release or SERCA, may be more dysfunctional in ischemic than non‐ischemic cardiomyopathy. This is the first evidence of dysfunctional Ca2+ influx in human atherosclerotic CSM.
U46619‐induced contraction is impaired by COX activity in coronary arteries from renal hypertensive rats
Authors: de Paula Tiago Dal‐Cin , Grando Marcella Daruge , Bendhack Lusiane Maria
Published: November 2023

Introduction

The coronary vascular bed is responsible for the proper myocardial blood supply and cardiac work. The vascular endothelium controls the vascular tone by the production and/or release of relaxing factors and contractile factors. In cardiovascular diseases like hypertension this balance can be impaired with increased prostanoids production like thromboxane derived from COX and TP receptors activation. Therefore, this work aimed to evaluate the contraction induced by the TP receptors agonist tromboxane analogue (U46619) in renal hypertensive (2K‐1C) and normotensive (2K) coronary arteries. The procedures were approved by the Ethics Committee: USP‐2015.1.441.60.7.

Methods

Hypertension was induced by surgery in rats. After six weeks, the rats were killed, the heart was removed and placed in ice cold Krebs solution to isolate the septal coronary arteries. The vessels were mounted in microvessels myograph and stimulated with 120 mM KCl and concentration‐effect curves were performed to 5‐hydroxytryptamine (5‐HT, 10 nM–100 μM) following the relaxation to acetylcholine (10 μM). In addition, concentration‐effect curves were constructed to U46619 (10 nM–10 μM) in the absence (Control) or in the presence of COX inhibitor (Indomethacin, 10 μM), selective COX inhibitors of COX‐1 (SC560, 10 μM) and COX‐2 (SC236, 10 μM). The coronary arteries were collected and frozen at −80° C to measure COX‐1 and COX‐2 expression. Data were analyzed by student's t test and one way ANOVA with Dunnet's post‐test (P<0.05).

Results

The coronary diameter (2K: 337.4 ± 29.9 vs 2K‐1C: 260.4 ± 27.6 μm), the contraction induced by KCl (2K: 4.28 ± 0.65 vs 2K‐1C: 2.98 ± 0.58 mN) and the acetylcholine‐induced relaxation (2K: 77.5 ± 6.5% vs 2K‐1C: 68.2 ± 14.0%) were not different. COX‐1 expression was not different between 2K and 2K‐1C rats (P=0.054). However, COX‐2 expression was higher in 2K‐1C than in 2K (P=0.02). Contraction‐induced by U46619 presented similar pD2 values in 2K (6.83 ± 0.17, n=5) and 2K‐1C (6.52 ± 0.10, n=6) but the maximum effect (ME) was lower in 2K‐1C (87.5 ± 17.0%) than in 2K (149.8 ± 13.1%). All the COX inhibitors used did not change the pD2 and ME in 2K coronary arteries. However, indomethacin increased the ME in 2K‐1C to 162.9 ± 16.9%, n=4 and SC560 increased the pD2 (7.33 ± 0.20, n=5).

Conclusion

Our results indicate that the contractile response induced by U46619 is impaired in 2K‐1C coronary arteries due to the production of relaxant factor derived from COX activity.

Support or Funding Information

Financial Support: CAPES, CNPq, FAPESP
Heparin Receptor Transmembrane Protein 184A (TMEM184A) Regulates Zebrafish Angiogenesis
Authors: Farwell Sara Lynn N , Reylander Kimberly G , Iovine M Kathryn , Lowe‐Krentz Linda J
Published: November 2023

Angiogenesis is the formation of new vasculature from existing vasculature and occurs normally during embryonic development, wound healing, and inflammation. Angiogenesis is tightly controlled by several regulators. Disruption of the balance between positive and negative regulators can result in pathological angiogenesis. Abnormal angiogenesis is implicated in several serious diseases, including cardiovascular disease, stroke, diabetes, cancers, and many others. Heparin and/or heparan sulfates (HS) play an important role during in angiogenesis by interacting with both proangiogenic and inhibitory factors. For example, heparin/HS has been shown to act as a scaffold between the major proangiogenic regulator vascular endothelial growth factor (VEGF) and its receptor (VEGFR2). We recently identified transmembrane protein 184A (TMEM184A) as the heparin receptor in vascular endothelial and smooth muscle cells. In vitro, TMEM184A specifically interacts with heparin/HS to mediate anti‐inflammatory and anti‐proliferative growth factor signaling in vascular cells. To examine the expression and function of the heparin receptor TMEM184A in vivo, we employed the zebrafish regenerating fin. We used the Tg(fli1:eGFP) transgenic line to easily visualize vascular regeneration. Immunofluorescence data show that TMEM184A is expressed specifically in endothelial cells in ontogenic and regenerating vasculature. TMEM184A colocalizes with GFP (the endothelial cells) in both arteries and veins in ontogenic fin rays, and TMEM184A expression matches the regenerating pattern of GFP over several days after amputation. In regenerating fins injected with heparin, we observe abnormal vascular regeneration characterized by incomplete vessels and a reduction in total and vascular regeneration length within 24 hours after injection. Remarkably, transient knockdown (≤ 24 hours) of TMEM184A using morpholino‐mediated strategies in regenerating fins results in a significant increase in endothelial cells in highly disorganized vasculature. These data are directly comparable to what we have reported in vitro. Our studies indicate that the heparin receptor TMEM184A plays a regulatory role during angiogenesis. It is important to continue to elucidate the mechanism(s) behind the function of TMEM184A and determine its potential for therapeutic targeting in pathological angiogenesis. Additionally, information detailing its function in vivo could be used to improve current heparin‐based therapies used in the prevention and control of cardiovascular disease and stroke.

Support or Funding Information

This work was supported by NIH grant HL536104 to LLK.
Acrolein‐Induced Epigenetic Modification of Histone 3 in Rat Vascular Smooth Muscle Cells
Authors: Esmaeili Mahsa , Philips Mathew Omana , Ranganna Kasturi , Joseph Mathew , Yousefipour Zivar
Published: November 2023

Objective

Environmental pollutants can exacerbate disease conditions by affecting chromatin structure and ultimately regulating gene expression. Acetylation of histone lysine has been reported to contribute to gene expression while the methylation results in gene inactivation. Acrolein, an environmental pollutant and a major component of cigarette smoke, has been implicated in cardiovascular toxicity and several neurological disorders such as Alzheimer's disease. N‐acetyl cysteine (NAC), a precursor of glutathione transferase, has been reported to prevent acrolein toxicity in cell culture. The goal of the present study was to investigate the effects of acrolein on modification of histone 3, lysine 9 residue (H3K9) in rat's vascular smooth muscle cells (VSMCs) in the present and absent of NAC.

Design and method

VSMCs were treated with 2 and 3 μg/ml of acrolein for 1, 6 and 24 hours in the present or absent of 0.2 mM NAC. At the end of the treatment, MTS essay was used to check cells viability. Western blot analysis was used to determine changes in H3K9 tri‐methylation and acetylation. Additionally, immunofluorescence staining was used to analysis cellular localization of H3K9 acetylation and methylation.

Results

Cells treated with 2 and 3 μg/ml of acrolein exhibited the highest toxicity at 6 hours incubation. Acrolein exposed VSMC displayed induction of histone H3K9 acetylation for all concentrations and time periods. The most significant induction of acetylation (30% at 2 μg/ml] and 46% [3 μg/ml]) and tri‐methylation (37% [2 μg/ml] & 65% [3 μg/ml]) occurred at 6 hours incubation. Co‐treated VSMCs with acrolein and N‐acetyl cysteine reduced H3K9 acetylation (100% for both 2 and 3 μg/ml), and tri‐methylation (115% & 125% for 2 and 3 μg/ml respectively). Immunofluorescence staining further confirmed changes in acetylation and tri‐methylation in acrolein treated VSCMs with or without NAC.

Conclusions

Based on our data we are concluding that effect of acrolein on VSCMs is partially due to alterations of H3K9 methylation and acetylation which can be prevented with addition of 0.2 mM NAC.

Support or Funding Information

NIH SC3GM103746‐03
Preserved Tolerance to Lower‐Body Negative Pressure during Heat Stress in Individuals with Multiple Sclerosis
Authors: Huang Mu , Allen Dustin R , Keller David M , Fadel Paul J , Frohman Elliot M , Davis Scott L
Published: November 2023

Multiple sclerosis (MS), a progressive neurological disease, can result in impairments in both the autonomic control of cardiovascular and thermoregulatory function. We tested the following hypotheses: (i) patients with MS (n=10) would exhibit diminished tolerance to a simulated hemorrhagic challenge compared to age, height, body weight, and sex matched healthy controls (CON, n=10) in normothermic, baseline conditions (NT, perfusing 34 °C water via tube‐lined suit) and (ii) this diminished tolerance would be exaggerated during a passive whole‐body heat stress (WBH, increased core temperature by 0.8 °C perfusing 48 °C water). Hemorrhage was simulated via progressive lower‐body negative pressure (LBNP) to presyncope and tolerance was quantified via a cumulative stress index (CSI). During NT, no differences were observed between MS and CON in response to LBNP (MS: 534±303, CON: 622±209 CSI; p=0.46). However, attenuated CSI reductions (% decline from NT) were revealed during WBH in MS compared to CON (MS: 29.9±23.4, CON: 58.6±21.8 % decline; p=0.01) suggesting a reduced impact of heat stress on LBNP tolerance in MS patients. One possible explanation for these findings is preserved fluid volume, as whole body sweat loss was lower in MS compared to CON (MS: 0.9±0.4, CON: 1.5±0.6 % body mass deficit; p=0.05). Contrary to our original hypotheses, these preliminary findings suggest LBNP tolerance was not different between groups at NT baseline and that individuals with MS had less of a decline in LBNP tolerance during WBH.

Support or Funding Information

Supported by NIH (R15HL11722401) and NMSS (RG4696A3/2) grants.
Prior Infection and Resultant Coagulopathy Increase Heat Stroke Severity
Authors: Dineen Shauna Marie , Plamper Mark L. , Ward Jermaine A. , Audet Gerald N , Leon Lisa R
Published: November 2023

Anecdotally, it has been suggested that prior viral infection increases heat stroke (HS) susceptibility and severity. HS and systemic infection have both been shown to induce disseminated intravascular coagulation (DIC), a condition associated with hypercoagulation, thrombocytopenia, multiorgan dysfunction, and higher mortality. We hypothesized that injection of the immunostimulant Polyinosinic: polycytidylic acid (PIC;100 μg) 48h or 72h prior to heat exposure (HE; 39.5±0.1°C) would exacerbate systemic inflammation and coagulopathy in a conscious C57BL/6J mouse model and increase HS morbidity. Prior to HE, mice injected with PIC showed significantly lower platelet counts at 48h (211 × 10^9/L) and 72h (324 × 10^9/L) compared to saline controls (604 × 10^9/L; P<0.05). In addition, PIC injected mice showed significantly elevated levels of D‐dimer (48h, 783212 pg/ml; 72h, 587265 pg/ml;P<0.05), thrombin‐antithrombin complexes (TAT) (48h, 8.18 ng/ml; P<0.05), thrombomodulin (48h, 28317 pg/ml; 72h, 28269 pg/ml; P<0.05), and decreased levels of antithrombin III (48h, 36527 ng/ml; P<0.05) compared to controls. These results indicate that prior to any HE, injection with PIC induced thrombocytopenia, coagulation, and fibrinolysis. Although PIC injected mice showed no outward signs of illness, HE 48h or 72h post injection was associated with increased mortality (30% and 10%, respectively), with survivors experiencing greater hypothermia depth (marker of HS severity; 29.5°C) compared to controls (30.9°C; P<0.01). HE also significantly increased D‐dimer levels (48h,953787 pg/ml; 72h, 864400 pg/ml; P<0.003) in PIC injected compared to mice exposed to either PIC or heat (601506 pg/ml) alone. Similarly, PIC plus heat induced significantly higher levels of thrombomodulin (48h, 32444 pg/ml; 72h, 33000pg/ml; P<0.02) than PIC or heat (28029 pg/ml) alone. Thus, PIC injection exacerbated systemic coagulation and fibrinolysis experienced during HE and was associated with increased HS severity and mortality. The thrombocytopenia observed in PIC injected mice may reflect a depression in platelet‐mediated immune function and a reduction in their overall capability to handle a second insult such as HE. Future studies will examine coagulation factors as potential biomarkers for predisposing conditions that increase HS susceptibility as well as any potential coagulopathy interventions or therapeutics to increase HS survival. Author views not official US Army or DOD policy.