bluuebunny/crossref_metadata_2025 huggingface dataset:
RAGE upregulation confers a proliferative phenotype to PANC‐1 pancreatic cancer cells
Authors: Swami Priyanka , Vetter Stefan W. , Leclerc Estelle
Published: November 2023

Pancreatic cancer is one of the deadliest cancer and is characterized by late diagnosis, aggressive clinical course and resistance to existing therapies. Recent efforts have been concentrated on identifying new targets and molecular pathways which contribute to the development and progression of this lethal disease, and which could be used to establish new therapeutic approaches.

Several reports have shown that the Receptor for Advanced Glycation End products (RAGE) contributes to the progression of pancreatic cancer. RAGE is demonstrated to be involved in processes like apoptosis, autophagy, and tumor cell bioenergetics. It is reported that RAGE is required for optimal mitochondrial function in tumors for ATP production. However, the effect of RAGE expression on the proliferative and migratory properties of pancreatic cancer cells is currently not known. A better understanding of the mechanisms of RAGE signaling could lead to the development of new therapeutic strategies in pancreatic cancer. Our objective was to investigate the effect of RAGE up‐regulation on the proliferation and migration properties of the PANC‐1 pancreatic cancer cell‐line. We generated cells that overexpress RAGE four‐fold compared to non‐transfected cells. We then compared the proliferation, migration and invasion properties of the new cells with those of the wild‐type cells. We also compared the signaling pathways affected by RAGE overexpression in these cells. We showed that four‐fold up‐regulation of RAGE in PANC‐1 cells resulted in increased cell proliferation to the expenses of cell migration. Both Boyden chamber and wound healing supported the reduced migratory properties of the RAGE overexpressing cells. Further analysis showed significantly reduced levels of integrins in the RAGE transfected cells. We suggest that in the PANC‐1 cells, which exhibit a mixed epithelial – mesenchymal phenotype, RAGE expression switches the phenotypic balance toward a proliferative phenotype. We are currently exploring how RAGE up‐regulation affects the expression of its ligands in these cells.

Support or Funding Information

This work is partially supported by the National Institutes of Health grant 1P20GM109024 and the NDSU College of Health Professions.
Induction of Autophagy and Apoptosis in Triple‐Negative Breast Cancer Cells by LT‐IIc, a Type II Heat‐Labile Enterotoxin
Authors: Heakal Yasser , Berlingeri Sofia Girald , Hu John , Federowicz Matthew , Connell Terry D , Masso‐Welch Patricia
Published: November 2023

Despite the recent advances in breast cancer treatment, triple negative breast cancer (TNBC) remains a serious health problem with poor prognosis and limited therapeutic options. Although hampered by development of resistance, chemotherapy‐based regimens remain the first‐line adjuvant choice for the treatment of TNBC. To combat development of resistance, it is essential to identify agents that induce cell death by independent induction of multiple cell death pathways. In this report, we screened cholera toxin (CT), a type I heat‐labile enterotoxin (HLT) of Vibrio cholerae, and LT‐IIa and LT‐IIb, type II HLT of Escherichia coli, for capacity to induce cell death specifically in triple‐negative MDA‐MB‐231 breast cancer cells. While CT, LT‐IIa and LT‐IIb had little effect on the cells, LT‐IIc reduced cell viability by ~50%, concomitant with development of extensive intracellular vacuolation. LT‐IIc‐induced cytotoxicity was not observed in MCF10A, a non‐tumorigenic breast epithelial cell line, or in MCF7, an estrogen receptor‐positive breast cancer cell line. LT‐IIc independently and simultaneously stimulated autophagy and apoptosis in MDA‐MB‐231 cells. Neither knockdown of ATG5, a key mediator of cellular autophagy, nor activation of caspase 3/7 disrupted LT‐IIc‐mediated cell death. Similarly, pharmacological disruption of autophagy using bafilomycin A1, did not interfere with LT‐IIc‐mediated cell death. In contrast, treatment of MDA‐MB‐231 cells with rapamycin, a known inducer of autophagy, enhanced LT‐IIc mediated cell death. Collectively, these findings indicate that LT‐IIc uniquely induces cell death through the divergent induction of autophagy and autophagy‐independent apoptotic pathways.

Support or Funding Information

D'Youville College School of Pharmacy and NIH
The β‐blockers carvedilol and nebivolol inhibit neoplastic transformation through a β2‐adrenergic receptor independent mechanism
Authors: Cleveland Kristan H , Chen Si , Guo Lei , Andresen Bradley T , Huang Ying
Published: November 2023

β‐adrenergic receptors (β‐ARs) are crucial mediators of physiological responses to catecholamines (i.e. epinephrine and norepinephrine), and β‐blockers competitively inhibiting β‐ARs thereby preventing the associated cardiovascular effects of β‐ARs. Aside from their cardiovascular function, β‐blockers are potential chemopreventive agents. Previous studies identified a subset of β‐blockers as potential cancer preventive agents. Carvedilol and nebivolol inhibit EGF‐induced neoplastic transformation of JB6 P+ cells, a non‐cancerous but transformation sensitive murine epidermal cell line. However, the antineoplastic mechanism(s) of action have yet to be elucidated. Therefore, we sought to evaluate the role of the β2‐AR, the only β‐AR present in JB6 P+ cells, in the β‐blocker antineoplastic effect. First, an EGF‐mediated soft agar transformation assay was utilized to evaluate the combined effect of carvedilol with either CGP 12177 or nadolol; CGP 12177 and nadolol lack a cancer preventive effect. 10 μM CGP 12177 (KD= −9.39) had no effect on carvedilol (KD= −9.40) mediated inhibition of EGF‐induced transformation of JB6 P+ cells: −6.48±0.09 IC50 versus −6.67±0.05 IC50 for carvedilol and carvedilol plus CGP 12177, respectively. Similarly, carvedilol combined with 10 μM nadolol (KD= −8.60) resulted in no shift in the concentration‐response curve (−6.55±0.03 IC50 versus −6.50±0.06 IC50). Since carvedilol is a non‐selective β‐AR and α1‐AR antagonist, we assessed the involvement of the α1‐AR by combining the β‐blocker nebivolol, that lacks α1‐AR activity, with 10 μM CGP 12177. Combination of nebivolol (KD= −7.91 for the β2‐AR) and CGP 12177 did not alter the potency of nebivolol (−6.46±0.11 and −6.81±0.12 for nebivolol and nebivolol plus CGP 12177, respectively). To further the pharmacological studies, the efficacy and potency of carvedilol in preventing EGF‐induced transformation of JB6 P+ cells was compared in scramble control shRNA and Adrb2 (β2‐AR) shRNA infected cells. With 72% β2‐AR knockdown, carvedilol prevented EGF‐mediated transformation of JB6 P+ cells (IC50= −6.02±0.07), although with a slightly reduced potency compared to the scramble control (IC50= −6.24±0.06). The data demonstrate that the β‐blocker carvedilol and likely nebivolol function as a cancer preventive agent independently of their widely‐understood action as β‐AR blockers.
Role of transient receptor potential canonical 5 (TRPC5) channels in vascular tone regulation
Authors: Liang Cai
Published: November 2023

Intracellular Ca2+ concentration in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) plays an important role in vascular tone control and blood pressure regulation. Previously, most research focused on voltage‐dependent Ca2+ channels which mediate Ca2+ influx and vascular contraction. However, recent focus has been shifted to TRP channels. Reports show that several TRP channels, including TRPC1 and TRPC3, have important regulatory role in vascular tone control. However, there are relatively few studies about the role of TRPC5 channels in vascular tone regulation.

In present study, we determined the expression of TRPC5 in mouse carotid arteries, aorta and mesenteric arteries. Immunohistochemical staining results demonstrated that TRPC5 is expressed in both ECs and VSMCs. Consistently, Western blot results also showed TRPC5 expression. Isometric tension of arteries was measured using wire myograph. In carotid arteries, selective TRPC5 inhibitor clemizole attenuated endothelium‐dependent contraction (EDC) in a dose‐dependent manner; another TRPC5 inhibitor ML204 also dramatically inhibited EDC. Importantly, EDC in carotid arteries from TRPC5 knockout (KO) mice was substantially lower compared with that of wild type (WT) mice. These results indicate that TRPC5 contributes to EDC response in mouse carotid arteries. Attempts were made to determine which cyclooxygenase (COX) mediates EDC in carotid arteries. In both TRPC5 WT and KO mice, selective COX‐2 inhibitor NS‐398 dramatically reduced EDC, while selective COX‐1 inhibitor VAS only slightly inhibited it. Non‐selective COX inhibitor indomethacin abolished EDC. Similar studies were performed using small mesenteric arteries. We found that ANG II‐induced contraction in KO mice was lower than in WT mice. This difference disappeared after removal of endothelium layer. Furthermore, clemizole significantly inhibited ANG II‐induced contraction. Taken together, our studies suggest that TRPC5 contributes to EDC response in mouse carotid arteries, and it also plays a role in ANG II‐induced contraction in mouse small mesenteric arteries.
Genetic Variation in 12‐Lox Regulates the Efficacy of ω‐3 and ω‐6 Fatty Acids to Suppress Platelet Activation
Authors: Shil Swarna , Hawley Megan , Li Cecilia , Holinstat Michael
Published: November 2023

Individuals with type 2 diabetes mellitus (T2DM), a metabolic disorder characterized by insulin resistance and hyperglycemia, are at an increased risk for a thrombotic event, the underlying pathology of strokes or heart attacks. Concurrently, T2DM patients are resistant to antiplatelet therapy, the current standard of care to reduce the morbidity and mortality associated with cardiovascular disease (CVD). Therefore, novel approaches to inhibit platelet activation are required to treat CVD in diabetics. One such approach is through the consumption of polyunsaturated fatty acids (PUFAs) – ω‐3 fatty acids (fish oils) or the ω‐6 fatty acid dihomo‐γ‐linolenic acid (DGLA). In platelets, 12(S)‐lipoxygenase (12‐LOX) is a key enzyme primarily responsible for the oxidation of PUFAs. Recent studies have shown that 12‐LOX was responsible for the anti‐platelet effects of DGLA. Interestingly, there have been 4 single‐nucleotide polymorphisms (SNPs) in the gene encoding 12‐LOX that impact its enzymatic activity. We hypothesize that these selective 12‐LOX SNPs will influence the ability of ω‐3 and ω‐6 fatty acids to suppress platelet activation. The activation of platelets isolated from diabetic (n=44) and healthy (n=45) patients following 2 months of PUFA supplementation was measured by RAP‐1 activation, integrin activation, calcium flux, and platelet aggregation to determine if genetic variants in 12‐LOX influenced the ability of PUFAs to inhibit platelet activation in diabetics versus healthy individuals. We found that there is a correlation between 12‐LOX genotype and platelet inhibition and a significant difference in diabetic versus healthy individuals.

Support or Funding Information

NIH HL114405
Novel Peptide Micellar Nanoformulation for Treatment of Thrombotic Complications of Sepsis
Authors: Bae Misuk , Rusu Luiza , Castellon Maricela , Du Xiaoping , Onyuksel Hayat , Minshall Richard D
Published: November 2023

Sepsis is one of the leading causes of death in hospital intensive care units in the U.S. Despite supportive care, microvascular thrombosis and disseminated intravascular coagulation in septic patients leads to ~40% mortality. Since there are no anti‐thrombotic drugs for sepsis on the market, new drugs are desperately needed. Interestingly, the plasma level of von Willebrand factor (vWF), the glue to which platelets adhere to form a clot, is elevated in septic patients and high levels predict poor outcome. We recently showed Gα12 is required for αSNAP‐dependent vWF secretion from Weibel Palade bodies. We identified a 6 aa sequence in the N‐terminus of Gα12, generated a cell permeable myristoylated alpha‐SNAP Binding Domain peptide, Myr‐SBD, and showed that this blocked thrombin and shear‐stress induced vWF secretion from human endothelial cells in vitro. In addition, intravenous delivery of Myr‐SBD to mice at the time of cecal ligation and puncture (CLP) surgery reduced vWF levels, decreased microvascular thrombosis in the kidney, and increased survival. Here, we extend these initial studies using a well‐characterized sterically‐stabilized simple micelle (SSM) system to generate a nanoformulation of Myr‐SBD peptide for in vivo delivery. Pilot pharmacokinetic and efficacy parameters of Myr‐SBD SSM were investigated by fluorescence spectrometry in vitro and in vivo. Fluorescent probe‐labeled Myr‐SBD (Myr‐SBD‐PDAM), prepared by modifying the carboxylic acid group of Myr‐SBD via PDAM conjugation was confirmed by fluorescence spectrometry. Myr‐SBD‐PDAM SSM was prepared by incubating the peptide with DSPE‐PEG2000 using a film‐rehydration method. Following SSM preparation, micelle size and morphology assessed by electron microscopy (TEM) and light scattering (DLS) revealed uniform micelles of 14.1 ± 2.7 nm in diameter. Uptake and intracellular accumulation of Myr‐SBD‐PDAM SSM was confirmed by fluorescence and confocal microscopy. Intravenous delivery of Myr‐SBD SSM to mice after CLP surgery improved survival compared to the untreated CLP group. Thus, nanoformulation of Myr‐SBD peptide may enable in vivo delivery for treatment of thrombotic complications of severe inflammatory response syndrome.

Support or Funding Information

This work was supported by MPI R01 HL125356 (RDM, XD), the Michael Reese Research and Education Foundation (RDM), and AHA (LR).
Apoptosis and autophagy in human vein grafts replacing occluded coronary arteries in atherosclerosis
Authors: Bortier Hilde Elisa , Kockx Mark , De Meyer Guido
Published: November 2023

Atherosclerosis is a chronic disease. For many decades vein grafts are used to replace occluded and suboccluded segments of coronary arteries. Restenosis is still a major problem today, as this chronic disease does not disappear when the occluded segments are replaced by autologous vein grafts. What are the cellular characteristics of this restenosis? Vein grafts obtained during surgical reinterventions were processed for light microscopy, SEM, TEM, immunohistochemistry (nuclear marker, smooth muscle cell marker, ..) and TUNEL (apoptosis).

The cellular characteristics are accumulation of foam cells at the luminal side and smooth muscle cell death in the layer underneath. The remaining smooth muscle cells show nuclear fragmentation or apoptosis and remnants of cytoplasmic fragmentation or autophagy. Attempts are being made to develop drugs that might prevent the accumulation of foam cells and smooth muscle cell death in the aim to prevent the redevelopment of atherosclerotic plaques in the vein grafts.
Microvascular Content and Insulin Sensitivity within Skeletal Muscle and Adipose Tissue Differs between Male and Female Mice on a High‐fat Diet
Authors: Abdifarkoosh Ghoncheh , Rudnicki Martina , Roudier Emilie , Haas Tara L.
Published: November 2023

Capillary networks within skeletal muscle and adipose tissue have a critical role in the delivery of energy sources and the maintenance of proper tissue function. A reduction in capillary density is correlated with impairments in whole body metabolic functions, as occurs in obesity. It is known that the susceptibility to develop obesity‐associated metabolic disturbances differs between males and females. However, studies investigating these sex‐related differences have largely overlooked the potential contribution of capillary content. The goal of this investigation is to determine whether there are sex differences in skeletal muscle and adipose tissue microvascular content and to relate these to insulin signaling in normal and obese mice. Male mice fed a high‐fat (HF) diet for 16 weeks exhibited reduced systemic insulin sensitivity compared to females who maintained a response similar to the normal‐chow (NC) group. Insulin stimulation increased levels of phosphorylated Akt (pAkt) protein equivalently in the skeletal muscle of male and female NC mice. With HF diet, males had significantly lower insulin‐stimulated pAkt levels vs. females (0.7 vs. 1.9, P<0.0001). Capillary‐to‐fiber ratio was not different in the skeletal muscle of HF male vs. female mice (1.5 vs. 1.4, n=3 ns). Ex vivo insulin stimulation of visceral adipose tissue increased pAkt in HF females but not in HF males. Microvascular content in visceral adipose was not different between NC males and females. In contrast, HF males had reduced, but HF females maintained, their adipose vascular content. This was further supported by higher mRNA levels of vascular marker CD31 (0.17 vs. 0.12, p<0.05) and pro‐angiogenic factor VEGF (0.14 vs. 0.07, p<0.05) in HF females vs. HF males. Our data suggest that capillary content and a pro‐angiogenic environment is maintained in the adipose of HF‐fed females, which in turn may be linked with maintained adipose insulin sensitivity.

Support or Funding Information

CIHR
Endothelial Cell Barrier Protein Expression Differs by Organ and Sex
Authors: Kemp Scott S. , Huxley Virginia H , Schramm Christine , Sieveking Steven W
Published: November 2023

In both humans and animals, while functional sex differences have been observed, the mechanisms responsible for these differences remains poorly understood. Hypotheses include exposure to reproductive hormones, DNA preprogramming, or a combination of the two. Our goal is to elucidate the molecular mechanisms responsible for observed sex differences in basal and activated barrier function of adult rat microvessels.

To this end we focused on cells isolated from age‐matched reproductively mature male and female rats (Sprague Dawley) to test the null hypothesis that protein expression does not differ by sex. Vascular cells were isolated from aorta and skeletal muscle to facilitate answering our question and compare our findings with previously published and observed data. Six markers associated with barrier function were chosen for study, CD 31 (PECAM‐1), αvβ3 (Integrin), N‐cadherin (CDH2), NCAM1 (CD 56), VCAM‐1 (CD 106), and ICAM‐1 (CD 54). Using flow cytometry we compared the difference in expression of these markers in sex‐verified (SRY gene expression) macro‐ and microvascular cells maintained in identical conditions of culture (Passage 8).

Our current data suggests sex differences in aortic EC barrier proteins: αvβ3, VCAM, NCAM and NCAD, with little to no sex difference observed in ICAM and CD 31. Sex differences have also been observed in the skeletal muscle EC barrier proteins: ICAM, VCAM, NCAM, with little to no difference observed in αvβ3, NCAD and CD 31. Organ differences have been observed between skeletal muscle and aortic EC barrier proteins in ICAM, VCAM, NCAM and NCAD, with little difference observed in αvβ3, and CD 31.

Given that the proteins forming the junctional complex and regulating adhesion to the extracellular matrix differ between males and females we possess an initial understanding of one mechanism whereby barrier function may vary by sex under basal conditions. Future studies will focus on the 2nd messenger pathways linked to these proteins and expression of proteins in these cells following exposure to vasoactive components shown to alter barrier function in a sexually dimorphic manner.

Support or Funding Information

NIH R01 DK095501
Capillary endothelial Gq protein‐coupled receptors and PIP2 toggle signaling between TRPV4 and Kir2 channels in the brain
Authors: Harraz Osama F , Longden Thomas A , Dabertrand Fabrice , Nelson Mark T
Published: November 2023

The ion channel composition of capillaries is not known. Using freshly dissociated capillary endothelial cells (cECs) from mouse brain, we measured currents through strong inward rectifier K+ (Kir2.1) channels and through transient receptor potential vanilloid (TRPV4) channels. We have proposed that Kir2.1 and TRPV4 channels are involved in electrical and calcium signaling, respectively, to transmit signals from cECs to upstream arterioles. We tested the hypothesis that these two channel types are functionally linked through phosphatidylinositol 4,5‐bisphosphate (PIP2). We found that intracellular PIP2 or ATP had opposite effects on TRPV4 and Kir2.1 currents, with PIP2 or ATP suppressing TRPV4 channel activity and conversely being essential for Kir2.1 activity. The level of PIP2 is controlled by the activity of Gq protein‐coupled receptors (GqPCRs), which hydrolyze PIP2 to produce inositol 1,4,5‐triphosphate (IP3) and diacyglycerol (DAG). We therefore tested the effects of prostaglandin E2 (PGE2; an agonist of the GqPCR EP1), which has been implicated in neurovascular coupling, on TRPV4 and Kir2.1 currents in cECs, with the expectation of divergent effects on the two conductances. Indeed, when measured simultaneously in the same cECs, PGE2 increased TRPV4 currents with half‐time (t0.5) of 3.5 minutes and decreased Kir2.1 currents with a t0.5 of 5.3 minutes. In conclusion, the present study supports the concept that PIP2 tonically inhibits TRPV4 channels and activates Kir2.1 channels in the brain capillary endothelium. GqPCR activation therefore acts as a molecular switch that alters the balance between electrical (Kir2.1) and Ca2+ (TRPV4) signaling, which might change the signaling modality to upstream penetrating arterioles, and would have profound effects on the control of blood flow into the brain.

Support or Funding Information

Supported by the NIH, Fondation Leducq and EC Horizon 2020 grants.
Signal Inference: A Method to Locate and Track Calcium Waves with Statistical Confidence
Authors: Billings Eric , Socha Matthew J , Domeier Timothy L , Balaban Robert S , Segal Steven S
Published: November 2023

Optical imaging studies of cell signaling pose a recurring challenge to track signals relative to the topology of the cell structures. Image analysis platforms require the user to visually assess which signals merit further study and define them as geometric regions of interest (ROIs) ‐ throwing away information outside the ROI. Tools for segmentation, the process of identifying cellular structures in an image, let the user subjectively identify biological structures with pixel resolution. Thus, the experimentalist is challenged to objectively control for observer bias. We have developed a method to avoid manual ROI generation and use the entire data set to extract the physiology of calcium (Ca2+) signaling within and between vascular endothelial cells. We hypothesize that signal tracking and segmentation could be made rigorous and semi‐automatic by requiring signals to be continuous in time and contiguous in space. This reduces the analysis of the pixel covariance matrix to tracking contiguous 2D foci with high covariance. We test if this is sufficient to 1) identify cell structure; 2) track Ca2+ signals; and 3) provide statistical confidence metrics. The algorithm was developed using data from confocal fluorescence imaging of cytosolic Ca2+ release and reuptake in freshly isolated endothelial cell tubes following local stimulation with acetylcholine (ACh) from a micropipette (1 μm tip, 1–2s). Results were tested with biological replicates under varied technical (scan rate, gain, amplification) and physiological conditions (stimulus dosage, location) and compared to manual segmentation and tracking. We illustrate a novel method for robust signal tracking with a minimum of user settings under different dose and gain settings. The method segmented cells by identifying foci with high spatial correlation. Individual foci overlap 78.9±18.1% of the area of manually segmented cell (n=5). Foci shape was also reproducible across different conditions (high/low gain for two ACh dosages). The foci boundaries for one preparation under four acquisition conditions were within 2.2 μm along 70.8% of the boundary (5 cells). This approach provided multiple metrics for each time course including the number, size, shape, rate and behavior of detected optical signals and cell structures. Typical run time for a single dataset (256 × 1024 pixels and 1054 frames) was less than 30 minutes on a (16GB RAM) computer.

Support or Funding Information

NIH F32HL107050, K01AG041208, R37HL041026
Increased propensity for cerebral microhemorrhages in a IGF‐1 deficient mouse model of accelerated vascular aging
Authors: Tarantini Stefano , Valcarcel‐Ares Marta Noa , Yabluchanskiy Andriy , Springo Zsolt , Fulop Gabor , Ashpole Nicole , Gautam Tripti , Giles Cory , Wren Jonathan D. , Sonntag William E. , Csiszar Anna , Ungvari Zoltan
Published: November 2023

Clinical and experimental studies show that aging exacerbates hypertension‐induced cerebral microhemorrhages (CMHs), which progressively impair neuronal function. There is growing evidence that aging promotes IGF‐1 deficiency, which compromises multiple aspects of cerebromicrovascular and brain health. To determine the role of IGF‐1 deficiency in the pathogenesis of CMHs, we induced hypertension in mice with liver‐specific knockdown of IGF‐1 (Igf1f/f + TBG‐Cre‐AAV8) and control mice by angiotensin‐II plus L‐NAME treatment. In IGF‐1 deficient mice the same level of hypertension led to significantly earlier onset and increased incidence and neurological consequences of CMHs, as compared to control mice, as shown by neurological examination, gait analysis and histological assessment of CMHs in serial brain sections. Previous studies showed that in aging increased oxidative stress‐mediated MMP activation importantly contributes to the pathogenesis of CMHs. Thus, it is significant that hypertension‐induced cerebrovascular oxidative stress and MMP activation were increased in IGF‐1 deficient mice. We found that IGF‐1 deficiency, by impairing hypertension‐induced adaptive media hypertrophy and extracellular matrix remodeling, also perturbs arteriolar remodeling processes, which act to restore circumferential stress, preventing mechanical damage to the vascular wall. Collectively, IGF‐1 deficiency promotes the pathogenesis CMHs, mimicking the aging phenotype, which likely contribute to its deleterious effect on cognitive function. Therapeutic strategies that up‐regulate IGF‐1 signaling in the cerebral vessels and/or reduce microvascular oxidative stress and MMP activation may be useful for the prevention of CMHs, protecting neurocognitive function in high‐risk elderly patients.

Support or Funding Information

This work was supported by grants from the American Heart Association, the National Center for Complementary and Alternative Medicine, and the National Institute on Aging
Spatio‐Temporal Analysis of Intracellular and Intercellular Calcium Signaling in Microvascular Endothelial Cell Tubes
Authors: Socha Matthew J , Billings Eric , Domeier Timothy L , Balaban Robert S , Segal Steven S
Published: November 2023

Intracellular Ca2+ concentration is an essential component of endothelial cell‐dependent regulation of vasomotor control of tissue blood flow. Coordination of vasodilation in microvascular resistance networks requires intercellular signaling in a direction opposite to blood flow to dilate upstream branches (e.g., feed arteries). We hypothesized that individual endothelial cells function both as elementary operating units of intracellular Ca2+ signaling and as elements of a syncytium in propagating intercellular Ca2+ signals. Intact endothelial cell tubes (n=3) were freshly isolated from superior epigastric arteries of male C57BL/6 mice (3–6 months), loaded with Ca2+ indicator dye (fluo‐4, 10 μM), and superfused continuously at 3 ml/min in a laminar flow stream of physiological salt solution (PSS, 25°C). The center of a tube was stimulated locally with acetylcholine (ACh) using microiontophoresis (1 μm tip, 800 nA, 2 s) to initiate Ca2+ signals that were monitored both upstream and downstream from the site of stimulation. Ca2+ responses were analyzed using a novel time series image analysis to define and track signals both within and across multiple cells. Individual cells behaved as operational units within the larger syncytium of the endothelium. Thus, individual cells exhibited intracellular Ca2+ signals that propagated longitudinally at 19.9±9.5 μm/s and laterally at 15.6±4.5 μm/s (P<0.05). Moreover, Ca2+ waves propagated from cell to cell along the intact endothelium at 23.4±0.7 μm/s in the upstream direction, opposite to the flow of PSS. In contrast, Ca2+ responses spread in the same direction as PSS flow (i.e., downstream) at > 100 μm/s, suggesting a combination of intercellular Ca2+ waves and convection of ACh. These findings resolve the ability of individual endothelial cells to generate bidirectional intracellular Ca2+ waves while engaging their neighbors in signaling as a functional syncytium along the vessel axis. The greater rate of Ca2+ wave propagation upstream along the intact endothelium vs the mean intracellular propagation rate suggests a role for feed‐forward signaling between cells that is readily distinguished from agonist convection and receptor activation occurring downstream from the site of initiating Ca2+ signaling. These data provide a reference for determining how vascular disease may affect endothelial Ca2+ signaling within and among cellular elements of the functional syncytium and thereby influence vasomotor control.

Support or Funding Information

NIH F32HL107050, K01AG041208, R37HL041026
Stromal cells promote neovascular invasion across tissue interfaces
Authors: Dale Jacob , Krishnan Laxminarayanan , Aliaj Klevis , Beare Jason , Weiss Jeff , Hoying James
Published: November 2023

Vascular connectivity between adjacent vessel beds within and between tissue compartments is an essential aspect to any successful neovascularization process. To establish new connections, growing neovessels must locate other vascular elements during angiogenesis, often crossing matrix and other tissue‐associated boundaries and interfaces. How growing neovessels traverse any tissue interface, whether part of the native tissue structure or secondary to a regenerative procedure, is not known. Using an experimental model of angiogenesis, wherein growing neovessels must interact with a 3D interstitial collagen matrix interface, we determined that matrix interfaces act as a barrier to neovessel growth acting to deflect neovessels along the interface. Computational modeling of the matrix biomechanics at the interface indicates that differences in collagen fibril density near and at the interface prevent the growing neovessel from penetrating the interface via durotaxis. Interestingly, a sub‐population of stromal cells, more specifically tissue‐resident macrophages, included with the neovessels facilitates neovessel interface invasion during angiogenesis through a VEGF‐A‐dependent process. Importantly, only stromal cells originating on the neovessel‐side of the interface but not the opposite side promote invasion. Also, ubiquitous addition of VEGF‐A in the absence of stromal cells does not promote interface invasion. Together, our findings indicate that stromal cells instruct or guide, via VEGF‐A, growing neovessels across a matrix interface which normally deflects them. Furthermore, given that the stromal cells need to be spatially oriented near the growing neovessels, it's likely that the stromal cells are establishing spatiotemporal gradients of VEGF‐A across the interface as the mechanism of guidance. Therefore, our findings indicate that vascularization of a tissue, via angiogenesis, depends on stromal cells to provide positional cues to the growing neovasculature and provide insight into how a microvasculature is organized within a tissue.
Contribution of kv7 channels to acetylcholine‐induced dilation in human adipose arterioles
Authors: Nishijima Yoshinori , Zhang David
Published: November 2023

Voltage‐gated K+ (KV) channels, especially shaker‐related KV1, serve as important regulators of vascular tone and reactivity. Emerging evidence suggests that KCNQ or KV7 channels may represent another subfamily of KV channels contributing to vascular tone regulation. Functional KV7 channels have been detected in vascular smooth muscle and endothelial cells of several vascular beds, although their significance in mediating physiological responses remains largely unclear. We investigated the contribution of KV7 channels to acetylcholine (ACh)‐induced dilation in ex vivo human adipose arterioles. Fresh adipose tissues were obtained as discarded surgical specimens from subjects without and with coronary artery disease (non‐CAD and CAD, respectively), and arterioles (100–200 μm, internal diameter) were dissected and cannulated under 60 mmHg and examined for diameter changes using video microscopy. In arterioles from non‐CAD subjects, ACh (10−9–10−5 M) induced potent vasodilation in a concentration‐dependent manner (% maximal dilation at 10−7 and 10−5 M, 62±6 and 93±2, respectively, n=6). KV7 channel blocker linopirdine (10 μM) significantly reduced the dilation to 10−7 M ACh (% dilation 18±6 vs. 62±6 in control), whereas blockade of KV1.5 (a major KV1 channel in human adipose arterioles) by DPO‐1 (1 μM) or Psora‐4 (30 nM) was without effect. In arterioles pretreated with L‐NAME (100 μM, a nitric oxide synthase inhibitor) and indomethacin (10 μM, a prostacyclin inhibitor), linopirdine also reduced ACh‐induced dilation (% dilation to 10−7 ACh, 10±4 vs. 30±6 in control with L‐NAME and indomethacin, and % dilation to 10−6 M ACh, 45±8 vs. 80±4 in control with L‐NAME and indomethacin). In contrast to arterioles from non‐CAD subjects, ACh‐elicited dilation in CAD arterioles was not affected by linopirdine (% dilation at 10−7 M, 57±4 vs. 66±7 in control). These results indicate that KV7 channels are involved in ACh‐induced dilation in human adipose arterioles from non‐CAD subjects and part of this effect is related to an endothelium‐dependent hyperpolarization (EDH) mechanism of dilation. In addition, the involvement of KV7 in ACh‐induced arteriolar dilation seems to be reduced in subjects with disease (CAD). This study provides further support for the recruitment of KV7 to the functional K+ channel repertoire in vascular cells.

Support or Funding Information

National Heart, Lung and Blood Institute Grant R01‐HL 096647
Vasodilatory Function in Human Skeletal Muscle Arteries with Advancing Age: Role of Adropin
Authors: Kwon Oh Sung , Andtbacka Robert , Hyngstrom John , Richardson Russell
Published: November 2023

Background

Aging is associated with impaired endothelium‐dependent dilation in human skeletal muscle feed arteries (SMFAs). However, the specific mechanisms underlying the age‐related impairment in the regulation of blood flow have yet to be clarified. Recent evidence has suggested that adropin, a peptide secreted by the liver, may play a role in cardiovascular health. Therefore, this study sought to determine if treatment with adropin, in vitro, can restore the age‐related endothelial dysfunction in human SMFAs.

Methods

Adropin protein expression was assessed in 3 groups of subjects, young (28±4 yrs, n=10), middle aged (50±6 yrs, n=10) and old (75±7 yrs, n=16) with Western blot analysis. Using pressure myography, vasodilation in SMFAs with and without adropin incubation, from these young, middle aged, and old subjects was assessed in response to three stimuli 1) flow‐induced shear stress, 2) acetylcholine (ACh), and 3) sodium nitroprusside (SNP).

Results

Progressive age‐related reductions in endothelial‐dependent vasodilatory function were evident in response to both flow (Young: 97 ± 13; Middle‐age: 62 ± 12; Old: 32 ± 14%, P < 0.05) and ACh (Young: 98 ± 15, Middle‐age: 68 ± 13 %; Old: 40 ± 10 %, P < 0.05). Adropin treatment in the middle aged and old SMFAs restored the vasodilatory response to flow (Middle‐age + Adropin: 88 ± 10; Old + Adropin: 78 ± 11 %) and ACh (Middle‐age + Adropin: 94 ± 16%; Old + Adropin: 83 ± 10 %, P < 0.05) (p<0.05). Endothelium‐independent vasodilation (SNP) was unaffected by age and adropin, also, had no effect. Interestingly, adropin upregulated eNOS protein expression in the middle aged and old SMFAS and there was a significant positive correlation between adropin protein expression and both flow‐mediated and ACh‐dependent vasodilation.

Conclusions

Overall, these findings suggest that adropin may play a critical role in age‐associated endothelial dysfunction. Furthermore, adropin could be a novel therapeutic approach to restore endothelial function and NO bioavailability in the elderly.

Support or Funding Information

NIH Heart, Lung, Blood Institute grants (HL‐ 091830) and Veterans Affairs Rehabilitation Research and Development Merit (E6910‐R)
Enhanced myogenic response of cerebral arteries induced by pre‐existing hypertension is intact after traumatic brain injury
Authors: Szarka Nikolett , Amrein Krisztina , Czeiter Endre , Buki Andras , Koller Akos , Toth Peter J
Published: November 2023

Traumatic brain injury (TBI) impairs pressure‐induced myogenic autoregulation of cerebral blood flow (CBF), which is associated with increased mortality and bad functional outcome of TBI patients. When blood pressure drops lack of myogenic autoregulatory function of cerebral vessels exposes brain tissue to hypoperfusion and ischemia; in case of high blood pressure it allows high blood volume to penetrate the brain exacerbating disruption of the blood brain barrier, development of hemorrhages and elevation of intracranial pressure. Hypertension, the most important cardiovascular risk factor in TBI patients, enhances myogenic tone of cerebral arteries via increased vascular production of 20‐hydroxyeicosatrienoic acid (HETE), which protects brain tissue form pressure/volume overload but leads to increased susceptibility to cerebral ischemia. Although both effects may potentiate the vascular consequences of TBI, it is not known how hypertension modulates the effect of TBI on myogenic responses of cerebral vessels. To establish the effect of TBI on cerebral myogenic responses in hypertension we studied isolated middle cerebral arteries (MCA) of normotensive and spontaneously hypertensive rats (SHR) after severe impact acceleration diffuse brain injury. We found that TBI diminished myogenic constriction of MCAs in normotensive animals, but the 20‐HETE mediated enhanced myogenic tone in SHRs was not affected by traumatic brain injury. These results suggest that following brain trauma hypertensive patients may be protected from high blood pressure, but are likely to be more prone to cerebral ischemia when blood pressure drops, and therefore require different therapeutic strategies than normotensive patients.

Support or Funding Information

This work was supported by grants from the Marie Curie Actions SMARTER 7th Framework Program of the European Union 606998 (to NSz and AK), the Hungarian Academy of Sciences (the Bolyai Research Scholarship BO/00634/15 to PT), the Hungarian Brain Research Program (Grant No. KTIA_13_NAP‐A‐II/8 to PT, ECz and AB) and the Hungarian Scientific Research Fund (K108444 to AK).
Arteriolar Vasodilation Involves Actin Depolymerization
Authors: Clifford Philip S. , Ferguson Brian S. , Jasperse Jeffrey L. , Hill Michael A.
Published: November 2023

It is generally assumed that relaxation of arteriolar vascular smooth muscle occurs through hyperpolarization of the cell membrane, reduction in intracellular calcium concentration, and activation of myosin light chain phosphatase/inactivation of myosin light chain kinase. We hypothesized that vasodilation is related to depolymerization of F‐actin. Cremaster muscles were dissected from rats under sodium pentobarbital anesthesia (50mg/kg). Cremaster 1A arterioles (n=20) were dissected free of surrounding skeletal muscle and cannulated with glass micropipettes after which they were pressurized and warmed to 34°C. Internal diameter was monitored with an electronic video caliper. The concentration of G‐actin was determined in flash frozen whole arterioles by ultracentrifugation and western blot. Arterioles dilated by > 40% of initial diameter in response to pinacidil (10−5mM) and sodium nitroprusside (5×10−5mM). The G‐actin/sm22α ratio was 1.43±0.18 at 10mmHg in arterioles with no tone and 0.67±0.09 at 70mmHg with myogenic tone. The G‐actin/sm22α ratio increased to 1.32±0.34 when arterioles were dilated with pinacidil and 1.14±0.18 with sodium nitroprusside. Since F and G‐actin are reciprocally related, the increase in G‐actin/sm22α indicates F‐actin depolymerization. These findings suggest that actin depolymerization is an important mechanism for vasodilation to external agonists.
CRISPR/Cas9 creation of a mouse lacking a unique motif on alpha globin demonstrates its critical role in regulating nitric oxide in vivo
Authors: Filho Gilson Bras Broseghini , Butcher Joshua T , Keller Steven TC , Poonam Sharma , Kwangseok Hong , DeLalio Leon J , Wenhao Xu , Padilha Alessandra Simão , Columbus Linda , Sonkusare Swapnil , Isakson Brant E
Published: November 2023

Alpha globin and eNOS form molecular complexes in microvascular endothelial cells that can regulate nitric oxide (NO) availability. On the alpha globin protein we have identified the amino acids 35–44 (FASFPTTKTY) as being essential for eNOS interaction. Using in silico prediction, we identified the 37–40 amino acid motif (SFPT) as being especially crucial for eNOS interaction, which we confirmed with purified protein binding assays. To confirm the importance of this motif in vivo, we used CRISPR/Cas9 to create a mouse with alpha globin missing amino acids 37–40 in the Hba1 gene. Whole genome sequencing confirmed the mutation and demonstrated a lack of any off‐target effects. We found that homozygous Hba1Δ37–40 mice were not viable (0 mice born from 8 different heterozygous pairs; 16 litters) and were re‐absorbed in utero by approximately day E12. The lethal homozygous Hba1Δ37–40 embryos had significantly dilated arteries and cardiac ventricles. For this reason we utilized Hba1WT/Δ37–40 heterozygous animals; these mice had no difference in total alpha globin protein expression in arterioles and were viable. These mice had decreased blood pressure and significantly increased heart rates. Angiotensin II infusion only had a mild effect on blood pressure that was significantly lower than controls with Angiotensin II. Blood NOX levels were significantly higher than controls. In line with this observation, third‐order mesenteric arterioles from heterozygous Hba1WT/Δ37–40 mice had significant increases in NO release when stimulated with the endothelial muscarinic agonists carbachol. We also detected a significant decrease in the amount of cGC present in smooth muscle cells. Our results demonstrate the importance of discreet motifs on alpha globin in regulating NO in endothelium in a unique CRISPR‐derived in vivo mouse model.

Support or Funding Information

Funding: HL088554,HL120840.
Maintenance of Nerves in the Rat Mesentery Culture Model
Authors: Hodges Nicholas
Published: November 2023

Neurovascular patterning represents an emerging area of microvascular research and is critical for tissue remodeling. While overlapping molecular signals highlight links between angiogenesis and neurogenesis, advancing our understanding of their spatial and temporal coordination is limited by a lack of in vitro models that contain both systems. One such potential model is the rat mesentery culture model, which our laboratory has recently introduced as an ex vivo tool to investigate cellular dynamics during angiogenesis and lymphangiogenesis in an intact microvascular network scenario. The objective of this study was to evaluate the effect of media type on nerve maintenance in the rat mesentery culture model. Adult male Wistar rat mesenteric tissue windows were harvested, rinsed in sterile DPBS and media and then cultured per media group: 1) minimum essential media (MEM) and 2) nerve basal medium (NBM) with NGF and 20% fetal bovine serum (FBS). On day 3 tissues were labeled for PECAM and class III β‐tubulin or PECAM and NG2. Unstimulated (not cultured) tissues were also labeled as a control group. Nerve presence at the arteriole level was quantified by the ratio of network feeding arterioles with nerve alignment to the total number of arterioles. Nerve presence at the capillary level was quantified by the length of nerve alignment per vascular area (μm/mm^2). NG2 and class III β‐tubulin positive nerve presence along feeding arterioles was decreased for MEM only group compared to control levels (NG2: Control = 0.875 ± 0.06, MEM = 0.6 ± 0.09, p < 0.05; Class III β‐tubulin: Control = 0.937 ± 0.06, MEM = 0.412 ± 0.10, p < 0.05). In contrast, nerve presence along arterioles identified by either label was not statistically different between the control and NBM+NGF+FBS groups (NG2: Control = 0.875 ± 0.06, NBM+NGF+FBS = 0.917 ± 0.05; Class III β‐tubulin: Control = 0.937 ± 0.06, NBM+NGF+FBS = 0.937 ± 0.06). NBM+NGF+FBS also maintained NG2 and class III β‐tubulin positive nerve length per vascular area compared to the control group (NG2: Control = 1930 ± 134, NBM+NGF+FBS = 1846 ± 214 μm/mm^2; Class III β‐tubulin: Control = 1134 ± 124, NBM+NGF+FBS = 1589 ± 299 μm/mm^2). Nerve presence at the capillary level was not quantified for the MEM group because of the lack of consistent capillary regions with observable nerves. The results of this study supports the use of specific media types to maintain nerve presence across cultured microvascular networks and implicates the rat mesentery culture model as a novel tool to investigate neurovascular patterning ex vivo.

Support or Funding Information

NIH R01AG049821, NIH P20GM103629