bluuebunny/crossref_metadata_2025 huggingface dataset:
Lef1‐mediated up‐regulation of Epac1 expression in Chronic Lymphocytic Leukemic Cells
Authors: Brown Loren , Murray Fiona , Zhang Lingzhi , Insel Paul A
Published: June 2021

Exchange protein activated by cAMP 1 (Epac1), a guanine‐nucleotide exchange factor for the small G‐protein Rap. Contributes to anti‐apoptosis and other cAMP‐mediated actions. Limited previous studies have noted that up‐regulation of Epac1 expression in chronic lymphocytic leukemia (CLL). We assessed peripheral blood mononuclear cells (PBMC) and found that Epac1 mRNA (real‐time PCR) and protein (immunoblotting) are up‐regulated ~70 fold and ~10 fold, respectively, in CLL patients compared to controls. We hypothesized that this increase in Epac1 expression is results from increased transcriptional activity of the Epac1 gene. Deletional analysis of the 2kb promoter region directly upstream of the Epac1 start site revealed that the region necessary for basal expression is located within 100 base pairs of the transcriptional start site. Overexpression of Lef‐1, a transcription factor up‐regulated in CLL, increased activity of a Epac1 promoter luciferase reporter in a CLL cell line but not a normal B‐cell cell line. Stimulation of CLL cells with Wnt3 did not increase activity of the Epac1 promoter reporter but increased activity of a Lef1 luciferase reporter. These results, which indicate that Epac1 is up‐regulated in CLL patients and that Lef1 regulates Epac1 by a non‐cannonical pathway that is unique to the CLL B cells, define a transcriptional mechanism for the regulation of Epac expression in human disease.
A Homogenous bioluminescent, and fast assay for monitoring alteration in cAMP cellular and tissue cAMP and monitoring the Modulation of Gs and Gi Protein Coupled Receptors
Authors: Goueli Said , Hsiao Kevin
Published: June 2021

G Protein Coupled Receptors represent a large validated target for Drug discovery research. The Gs is coupled to activation of adenylate cyclase and Gi is coupled to inhibition of adenylate cyclase while Gq is coupled to activation of phospholipase β. Cellular concentrations of second messengers such as cAMP and cGMP have been known to play major roles in smooth muscle relaxation, contractile activity of cardiovascular system, and central nervous system related pathologies. We report here on a new assay for determining cAMP concentrations in cellular and tissue extract and for monitoring modulation of GPCR that are linked to activation or inhibition of adenylate cyclase (Gs or Gi). The assay is homogenous, fast, HTS formatted with high Z′, and adaptable to multi plate formats. We have successfully generated EC50 values for agonists and IC50 values for antagonists of Gs coupled receptors that are similar to those reported in the literature. The assay is easy to use, and can be carried out in less than 60 minutes and does not require antibodies or expensive instrumentation for signal detection and the signal output is relatively stable for few hours.
Activation of G protein‐coupled estrogen receptor 1 (GPER) induces coronary artery relaxation via cAMP/PKA pathway
Authors: Yu Xuan , Li Fen , White Richard , Stallone John , Han Guichun
Published: June 2021

Activation of the membrane estrogen receptor, GPER, may lower high blood pressure and preserve cardiac function after reperfusion. We reported that GPER activation induces a rapid relaxation of porcine coronary arteries by opening of BKCa channels. However, downstream signaling events in this pathway are poorly understood. The objective of our study was to investigate the mechanism of GPER‐mediated coronary artery relaxation. We found that the adenylyl cyclase (AC) inhibitor, SQ‐22538 (100 μM), attenuated G‐1 (GPER agonist)‐induced relaxation of pre‐constricted porcine coronary arteries, and shifted the G‐1 concentration‐relaxation curve to the left. These findings suggest that AC plays functional role in GPER action. Furthermore, G‐1 (1μM) stimulated cAMP production, and this stimulation was blocked by 5 μM G15 (GPER selective antagonist). Treating arteries with 5μM Rp‐8CPT‐cAMP (PKA selective inhibitor) significantly inhibited the G‐1 relaxation effect, and shifted the concentration‐response curve to the left. Lastly, 100 μM FMP‐API‐1 (an inhibitor of the interaction between PKA and the A kinase anchoring protein, AKAP) attenuated the relaxation effect of G‐1. In conclusion, G‐1 induces porcine coronary artery relaxation via cAMP/PKA signaling, which involves subcellular localization of PKA via interaction with AKAP.
Role of Beta2‐Adrenoceptor Signaling and Mitogen Activated Protein Kinases in IL‐13 Induced Mucus Production in Human Airway Epithelial Cells
Authors: Al-Sawalha Nour , Pokkunuri Indira , Omoluabi Ozozoma , Bond Richard , Knoll Brian
Published: June 2021

Excessive airway mucus production by airway epithelium is a hallmark feature of asthma that contributes to airway obstruction. Our objectives were to determine the requirements for β2‐adrenoceptor (β2AR) signaling and mitogen activated protein kinases (MAPKs) in enhancing the expression of MUC5AC, which encodes a major airway mucin, in response to IL‐13 in normal human bronchial epithelial cells (NHBECs). Cells were cultured at air‐liquid interface for 14 days on Transwell filter supports and MUC5AC expression was assessed by quantitative RT‐PCR. Only in the presence of epinephrine (3 μM) was MUC5AC transcription induced in response to IL‐13 (20 ng/ml); there was a 14.9‐fold increase in MUC5AC transcript numbers in cells cultured with IL‐13 and epinephrine compared to epinephrine alone. The addition of ICI‐118,551, a selective β2AR inverse agonist, blocked MUC5AC expression while CGP‐20712A, which is a β1AR selective antagonist, did not. MUC5AC expression in response to IL‐13 and epinephrine was blocked by treatment with chemical inhibitors of MEK‐1/2, ERK‐1/2, JNK or p38. We conclude that β2AR signaling is required to induce MUC5AC expression in response to IL‐13. Moreover, MAPKs are components of the required β2AR/IL‐13 receptor signaling pathways in producing MUC5AC expression in human airway epithelial cells.

Supported by NIH 1R01A179236 to RAB
Opening of TRPV4 channels induce relaxation mediated by KCa3.1 channels and nitric oxide synthase in mouse pulmonary arteries
Authors: Dalsgaard Thomas , Bajoriunas Vytis Bajoriunas , Köhler Ralf , Simonsen Ulf
Published: June 2021

This study investigated the synergistic actions of TRPV4 and KCa3.1 channels in relaxation of mouse pulmonary arteries. In wild type (WT) and KCa3.1−/− pulmonary arteries mice, TRPV4 channel currents were investigated by patch clamping, TRPV4 mRNA by qPCR, and isometric tension recordings in myographs. Opening TRPV4 channels with GSK1016790A (GSK) increased membrane current sensitive to HC067047, a blocker of TRPV4, in WT and KCa3.1−/− mice. TRAM‐34 decreased co‐activated KCa3.1 currents in WT mice. TRPV4 mRNA was similar in WT and KCa3.1−/− mice. ACh (10 μM) and GSK (10 nM) induced endothelium‐dependent relaxation that was reduced by NO synthase (NOS) inhibition, blocking TRPV4‐, and KCa3.1 channels in WT mice. In KCa3.1−/− mice, ACh‐ and GSK‐ induced relaxation was almost abolished. TRPV4 channel activation induced relaxation that required activation of KCa3.1 channels and NOS. Surprisingly, deficiency of KCa3.1 abolished relaxation to ACh and to TRPV4 activation. These results suggest that TRPV4 activation produces calcium influx resulting in KCa3.1 co‐activation as an essential step for endothelium‐dependent relaxation in pulmonary arteries.
Thyroid hormone affects both endothelial and vascular smooth muscle cells in rat arteries
Authors: Cai Yin , Leung George P.H. , Vanhoutte Paul M.
Published: June 2021

SAR, China, Peopleˈs Republic of The effect of thyroid hormone on vascular cells is controversial. The present study investigated in isolated arteries [with and without endothelium; suspended for isometric tension measurement] whether or not thyroid hormone causes relaxations in, or alleviates endothelial dysfunction in diabetic animals.

Triiodothyronine, (T3) did not relax aortae of control rats with or without endothelium but caused endothelium‐independent decreases in tension in mesenteric arteries, suggesting an endothelium‐independent vasodilator effect of the hormone.

In femoral arteries of rats with streptozotocin‐induced Type 1 diabetes, A23187 caused the release of endothelium‐derived contracting factors (EDCF). After four weeks of chronic treatment with T3, the contractions to A23187 of preparations with endothelium were unchanged, but those without endothelium were augmented, suggesting that the release of EDCF was reduced. Thus, thyroid hormone affects both endothelial and vascular smooth muscle cells in rat arteries.
Date palm fruits grown in Oman ameliorate amyloid beta protein induced cytotoxicity
Authors: Musthafa Mohamed Essa , Al-Adawi Samir , Al-Asmi Abdullah , Vaishnav Ragini , Gilles Guillemin J , Ramachandiran Nandagopal
Published: June 2021

Amyloid‐beta‐protein (Aβ) is a protein that clusters into amyloid plaques on the blood vessels and on the outside surface of neurons and ultimately leads to neuronal death. Such mechanisms have been postulated to be critical involved in the pathogenesis of Alzheimer's disease (AD). Evidences suggest that Aβ heighten free radical production in nerve cells which trigger cell death due to macromolecular oxidations and associated DNA fragmentation. Recently, considerable attention has been focused on a broad range of dietary antioxidants that have been implicated in scavenging free radicals, a process that is likely to be essential for mitigating oxidative stress that are thought to be strongly associated into the development of AD. In consonant with such view, we preliminary data indicate that date palm fruits grown in Oman promote solubility and inhibit in vitro fibrillation of Aβ peptides. The current study aims to analyze the effects of Omani date palm (Fardh variety) on Aβ‐induced cytotoxicity in rat pheochromocytoma (PC 12) cells. The present data suggest that Omani date palm fruits have potential to offer protection against Aβ‐induced cytotoxicity in a dose‐dependent manner, possibly by enhancing the endogenous antioxidant mechanism. The underlying biological process for such benefit has yet to be charted and therefore further research are warranted. This was funded by TRC Oman (RC/AGR/FOOD/11/01). COI:None
A novel neuroprotective curcuminoid alleviates glucose intolerance and improves insulin signaling
Authors: Panzhinskiy Evgeniy , Lapchak Paul A , Ren Jun , Sreejayan Nair
Published: June 2021

Type‐2 diabetes is growing at epidemic proportions and pharmacological interventions are being actively sought. This study examined the effect of a novel neuroprotective curcuminoid, CNB‐001 [4‐((1E)‐2‐(5‐(4‐hydroxy‐3‐methoxystyryl‐)‐1‐phenyl‐1H‐pyrazoyl‐3‐yl)vinyl)‐2‐methoxy‐phenol), on glucose intolerance and insulin signaling in high fat diet (HFD)‐fed mice. C57BL6 mice (5–6 week old) were randomly assigned to receive either a HFD (45% fat) or a low fat diet (LFD, 10% fat) for 24‐weeks, together with CNB‐001 (40mg/kg/d, ip). Glucose tolerance test revealed that that the area under the curve of post‐challenge glucose concentration was elevated on HF‐feeding, which was attenuated by CNB‐001. CNB‐001 attenuated body weight gain, serum triglycerides and IL‐6, augmented insulin signaling (elevated p‐Akt, p‐IRβ, lowered ER‐stress, PTP1B) and glucose uptake in gastrocnemius muscle of HFD‐fed mice. Respiratory quotient, measured using metabolic chamber, was elevated in HF‐fed mice, which was unaltered by CNB‐001, although CNB‐001 treatment resulted in higher energy expenditure. In cultured myotubes, CNB‐001 reversed palmitate‐induced impairment of insulin signaling and glucose‐uptake. Taken together, CNB‐001 alleviates obesity‐induced glucose intolerance and represents a potential candidate for further development as an antidiabetic agent. Supported by Wyoming INBRE (P20RR016474).
Histone deacetylation is the primary epigenetic mechanism for silencing of tumor suppressor gene ‐ Tissue Factor Pathway Inhibitor‐2 in hepatocellular carcinoma cells
Authors: Moghe Akshata , Barker David , Ghare Smita , Gobejishvili Leila , Joshi-Barve Swati , Barve Ashutosh , McClain Craig , Barve Shirish
Published: June 2021

Tissue factor pathway inhibitor‐2 (TFPI‐2) is a critical tumor suppressor gene that is frequently silenced in primary hepatocellular carcinoma (HCC). Inactivation of TFPI‐2 is associated with increased tumor growth and metastasis in HCC. We investigated the role of epigenetic regulation in TFPI‐2 expression using 5‐azacytidine (5‐AZA, a DNA methyltransferase inhibitor) and trichostatin A (TSA, a histone deacetylase inhibitor) in HCC cell lines. Both 5‐AZA and TSA upregulated TFPI‐2 expression; however, TSA‐mediated reactivation of TFPI‐2 was more robust, suggesting that histone deacetylation may be the predominant mechanism of TFPI‐2 silencing. In further studies, TSA greatly increased the acetylation of histones at the TFPI‐2 promoter region, as well as the binding of relevant transcription factors and RNA polymerase II at the transcription start site for TFPI‐2. Co‐treatment with Garcinol, a histone acetyltransferase inhibitor, substantially reduced the TSA‐induced upregulation of TFPI‐2, implying that the histone acetyltransferase‐histone deacetylase (HAT‐HDAC) balance critically regulates TFPI‐2 expression in HCC cells. Overall, our data suggest that histone deacetylation is the likely primary mechanism underlying the silencing of TFPI‐2 in HCC. HDAC inhibitors such as TSA are thus, potential chemotherapeutic agents in HCC. Supported by HRSA‐C76HF19368(SB).
Increased Lymphatic Permeability During Shock and Burn Trauma Alters Antigen Presenting Cell Recruitment to Mesenteric Lymph Vessels
Authors: Cromer Walter Emerson , Zawieja Scott , Stagg Hayden , Hunter Felicia , Tharakan Binu , Meininger Cynthia , Childs Ed , Zawieja David
Published: June 2021

Recent attempts to define mediators of lymphatic permeability and their functional roles have been made but few definitive conclusions have been drawn. We hypothesize that failure of the lymphatic barrier induced by inflammatory cytokines during trauma leads to enhanced trafficking of antigen presenting cells to the lymphatic in the mesentery.

Lymphatic endothelial cell (LEC) permeability was measured using a transwell FITC‐BSA assay. LECs were treated with inflammatory cytokines or serum from rats subjected to either hypovolemic shock (HVS) or burn trauma (BT). Also, mesenteric tissues were harvested from sham, HVS and BT and stained for MHCII expression.

LEC permeability increased when treated with HVS, or BT serum or inflammatory cytokines (in particular IL‐6, which produced responses similar to HVS & BT). Numbers of MHCII cells associated with mesenteric lymphatics increased with both HVS and BT as expected.

We examined the effect of mediators of LEC permeability and what effects this might have on immune cell trafficking. We conclude that LECs can alter their barrier in response to mediators of permeability and that the number of MHCII cells increases around lymphatics under conditions of increased permeability. Together these findings present a new role for lymphatics in both immunology and physiology.
Soluble guanylate cyclase activation protects against postischemic inflammation and reduces nitrate tolerance in heme‐oxygenase‐1 knockout mice
Authors: Wang Walter Zhuo , Jones Allan , Wang Meifang , Zuidema Mozow , Durante William , Korthuis Ronald
Published: June 2021

In previous studies, we showed that unlike wild‐type animals, heme oxygenase‐1 knockout mice (HO‐1 (−/−)) demonstrated nitrate tolerance and were not protected from the inflammatory effects of ischemia/reperfusion (IR) when preconditioned with hydrogen sulfide or ethanol, stimuli that induce downstream signaling by soluble guanylate cyclase (sGC)‐dependent mechanisms. BAY 60‐2770 activates oxidized sGC while BAY 41‐2272 stimulates reduced sGC (JPET 335:85‐91, 2010). We hypothesized that pretreatment with either BAY compound would limit postischemic inflammation and reverse nitrate tolerance in HO‐1(−/−) mice. Intravital fluorescence microscopy was used to visualize leukocyte rolling (LR) and adhesion (LA) in single postcapillary venules of the small intestine. Vasodilator responses to acetylcholine (ACH) or nitroprusside (NP) were measured on superior mesenteric arterial (SMA) rings isolated after IR and from Sham IR mice. Preconditioning with either BAY compound 10 min before or 24 h prior to IR reduced postischemic LR and LA in both WT and HO‐1(−/−), and increased SMA responses to ACH and NP in HO‐1(−/−). It is concluded that preconditioning with the sGC active agents, BAY 60‐2770 and BAY 41‐2272, limits postischemic inflammation and reduces nitrate tolerance in HO‐1(−/−). Supported by grants from the NIH (AA‐014945, HL‐095486 & HL‐59976). BAY 60‐2770 was a gift from Bayer Schering Pharma AG.
Collateral Artery Blood Flow Measurements Made in the Mouse Ischemic Hindlimb Model Using Deep Laser Speckle Imaging
Authors: Niu Jacqueline , Sumer Suna , Meisner Joshua K , Price Richard J
Published: June 2021

Laser speckle imaging (LSI) is a technique that measures relative blood flow in large vascular networks through the motion of scattered coherent light. Conventionally, LSI uses epi‐illumination, which collects the light scattering information from the surface and limits the depth of imaging to the most superficial tissues. We hypothesized that the use of transmitted coherent light could (1) project the dominant scattering signals from structures deeper in the tissue and (2) allow for in vivo measurements of blood flow in the superficial muscles of the mouse hindlimb during femoral arterial ligation (FAL). (1) In vitro testing in a muscle tissue phantom showed an ~3.4±0.3 fold increase in the flow signal at various depths from 0.6mm to 1.6mm and extension of the minimum signal depth by ~200%. (2) In vivo, we measured changes in blood flow through the two collateral arteries (CA) of the gracilis muscle in C57Bl6 mice. Hindlimbs were trans‐illuminated with a 658nm wavelength laser, and images were acquired before FAL, and 10, 20, and 30 minutes after FAL. Before FAL, flow in the CA was a function of longitudinal position: flow was high where CA entered the muscle and ~0 at the convergence point in the center of the muscle. After FAL, CA flows increased substantially and equilibrated along the vessel length. The concept of transmitted LSI allows for deep tissue imaging, and these data represent the first quantitative measurements of flow change throughout a CA network during mouse FAL, the standard model of revascularization research. NIH HL74082 and AHA 10GRNT3490001.
The effects of modulating eNOS activity and coupling on leukocyte‐endothelial interactions in rat mesenteric postcapillary venules
Authors: Koon Amber , Kern Maria , Young Lindon H , Rueter Brian , Iames Edward , Chen Qian
Published: June 2021

Leukocyte‐endothelial interactions associated with vascular injury are attenuated by endothelial‐derived nitric oxide (NO). Endothelial NO synthase (eNOS) in the presence of tetrahydrobiopterin (BH4) produces NO from L‐arginine and is termed eNOS coupling. However, when the ratio of dihydrobiopterin (BH2) to BH4 is increased, eNOS becomes uncoupled and produces superoxide instead of NO. Protein kinase C epsilon (PKC ε) positively regulates eNOS activity. This study examined modulating eNOS activity and coupling by superfusing BH2 (100 μM) by itself, combined with PKC ε activator (10μM) or PKC ε inhibitor (10μM), or combined with BH4 (100μM) and PKC ε activator in rat mesenteric venules. Intravital microscopy was used to evaluate leukocyte endothelial interactions within a 2 hr period. We found that BH2 (100μM n=3, P<0.05) or combined with PKC ε activator (n=5) significantly increased leukocyte rolling, adherence, and transmigration, compared to Krebs’ buffer controls (n=4, P<0.05). By contrast, the combination of PKC ε activator and BH4 significantly attenuated (n=5, P<0.05) BH2 induced leukocyte rolling/adherence. The BH2 induced response on all three leukocyte endothelial interactions was significantly attenuated by PKC ε inhibitor (n=4 P<0.05). The data suggest that promoting eNOS coupling or inhibiting eNOS uncoupling may be important mechanisms mediating inflammation induced vascular injury. .
High fat feeding leads to inflammation and depressed markers of immune system reactivity in rats
Authors: Liss Tyler , Reiss Rebecca A , Sweazea Karen L
Published: June 2021

Increased adiposity is strongly associated with inflammation. Prior studies have shown that feeding rats a high fat diet (HFD) leads to increased adiposity and inflammation, including elevated circulating tumor necrosis factor alpha (TNFα). The current study was designed to further examine whether markers of inflammation and immune system activation are altered in HFD rats. Rats were fed either a normal chow or HFD for 6 weeks. Comparative whole transcriptome sequencing of isolated aorta from HFD and chow fed rats indicates a significant (p<0.05) 2.8‐fold decrease in the expression of interferon regulatory factor 4 (IRF4), a factor required for lymphocyte activation, suggesting the immune system of these animals is downregulated. Results from western blot analyses show no difference in the expression of the anti‐inflammatory heat shock proteins 60 or 70 (HSP60/70) in erythrocytes or plasma from HFD rats compared to rats fed normal chow (p>0.05). Unlike TNFα, urinary levels of the inflammatory mediator prostaglandin E2 are not different between HFD and chow rats (p>0.05). These findings suggest that, despite elevations in TNFα, the immune system of HFD rats may be partly downregulated. Further studies testing the agglutination response of plasma samples collected from HFD and chow rats will help to assess how well circulating leukocytes respond to immune challenges in overweight animals.
The melanocortin receptor system in mediating anti‐inflammatory protection following cerebral ischemia reperfusion injury
Authors: holloway paul M , getting stephen j , gavins felicity
Published: June 2021

The melanocortin receptor system (MRS) provides an attractive target for the treatment of stroke, due to their ability to initiate anti‐inflammatory and neuroprotective pathways. However, the relative contribution of the receptors that mediate these effects (MC1 MC3 and MC4) have yet to be determined. This current work aims to assess the therapeutic potential of targeting the MRS for treatment of stroke.

The bilateral common carotid artery occlusion (BCCAo) mouse model of global stroke was utilised in conjunction with intravital microscopy, to quantify the neuro‐inflammatory response through real time visualisation of leukocyte recruitment in the cerebral microcirculation.

BCCAo induced significant increases in leukocyte rolling and adhesion in the cerebral microcirculation. Treatment with the pan receptor agonist α‐MSH significantly reduced the ischemia‐reperfusion induced leukocyte recruitment. Antagonism of MC3 and MC4 using SHU9119 failed to abrogate the anti‐inflammatory effects of α‐MSH early in reperfusion (40 min), but was able to blunt the effects of α‐MSH by 2h.

These results suggest that in the early stages of reperfusion, MC1 activation may be of particular importance in mediating protection within the cerebral microcirculation. However as the inflammatory reaction progresses the role of MC3 may become more prominent. This work was funded by the British Heart Foundation
Seasonal variation in thrombotic and thrombolytic factors in human plasma
Authors: Murray Erin Patrick , Field Kristina A. , Field Margaret F.
Published: June 2021

Thrombotic diseases have been shown to increase in the winter. This study sought to examine four thrombotic factors; Fibrinogen (Fb), Factor VII (FVII), Factor X (FX) and Tissue Plasminogen Activator Inhibitor (TPA‐I), and two thrombolytic factors Antithrombin III (ATIII) and Tissue Plasminogen Activator (TPA) in human plasma during winter and summer. To date, no comprehensive studies exist that look at these clotting factors simultaneously. Sandwich ELISA was used to measure the concentration of each factor from individual subjects. Statistical significance was found in seasonal variation of Fb, FVII, FX and TPA‐I (limited to females). A lower concentration of Fibrinogen was found in the winter, which was unexpected, but consistent with other studies. FVII increased in the winter, consistent with our original hypothesis. FX showed lower concentrations in the winter, which has not been previously reported in the literature. TPA‐I showed a marked increase in the summer but this increase was limited to females over 40. Neither TPA, nor ATIII showed significant seasonal variation. We conclude from this that a number of thrombotic factors increase in the winter but this is not consistent among all factors measured. Thrombolytic factors were unchanged. This research was supported by the Saint Mary's College of California Faculty‐Alumni Grant, Faculty Development Grant, and the Summer Research Program
Theoretical simulation of angiogenesis and structural adaptation in microvascular networks
Authors: Secomb Timothy W. , Alberding Jonathan P. , Dewhirst Mark W. , Pries Axel R.
Published: June 2021

A theoretical model is presented for simulating growth, structural adaptation and pruning of microvascular networks. The network is described as a set of interconnected segments. Blood flow is simulated using a hemodynamic model. Diffusion of oxygen and a growth factor in the extravascular space is simulated. Vessels are assumed to undergo structural diameter change, including pruning, in response to hemodynamic and metabolic signals, and angiogenesis is governed by growth factor levels. The model shows that over‐abundant stochastic angiogenesis occurring in parallel with refinement of the network by structural adaptation and pruning can generate networks that provide efficient convective transport over large distances, together with dense space‐filling meshes for short diffusion distances to every point in the tissue. Angiogenesis in response to a growth factor produced in hypoxic regions, and structural adaptation in response to both local and upstream propagated signals, are essential components of the model. This approach gives a framework for understanding vascular network formation in normal and pathological conditions and for predicting effects of therapies targeting angiogenesis. Supported by NIH grants HL034555 and CA40355.
Endothelial signaling of matrix‐bound and diffusible VEGF
Authors: Logsdon Elizabeth A. , Woo Laura , Mac Gabhann Feilim
Published: June 2021

Angiogenesis is a complex morphogenesis process coordinating endothelial cell (EC) signaling through matrix/integrin pathways and growth factor/receptor pathways, including the vascular endothelial growth factor (VEGF) family and their receptors. VEGF is presented to ECs in two modes – freely diffusing or bound to the extracellular matrix (ECM). While most studies have focused on diffusible VEGF, matrix‐bound VEGF also induces receptor activation resulting in unique signaling. Using a combination of experimental and computational methods, we examine how fibronectin, a component of the ECM known to bind VEGF, influences EC responses. Receptor activation, trafficking, and intracellular signaling are predicted to be highly altered for matrix‐binding VEGF. We independently control VEGF presentation mode and matrix‐integrin signals in vitro. Results indicate that diffusible VEGF is a stronger signal for VEGF receptor and adhesion molecule expression. Human microvascular ECs exhibit starkly different morphology based on the ratio of matrix‐bound to diffusible VEGF, with increased cell‐cell contact for diffusible VEGF. Thus, VEGF presentation mode controls EC behavior. Supported by NIH R00HL093219
Suppression of Vascular Network Formation by Human Cardiac Endothelial Cells with Low Concentrations of Doxorubicin is Due to the Inhibition of Endothelial Cell Proliferation
Authors: Konorev Eugene , Sun Zuyue
Published: June 2021

Anticancer drug doxorubicin (DOX) is known to cause dilated cardiomyopathy in treated patients. We set out to examine if cardiac endothelial cells present a target of doxorubicin in the heart. Primary human cardiac microvascular endothelial cells (HCMVEC) form vascular network in vitro when co‐cultured with human cardiac fibroblasts (HCFB). DOX suppressed the formation of vascular networks in a co‐culture assay with an IC50 of 8 nM. DOX did not inhibit the production of VEGF by these cells at these low concentrations under both normoxic and hypoxic conditions, but caused moderate inhibition of VEGF release by hypoxic HCFB at concentrations above 250 nM. Similarly, it did not enhance apoptosis in these cells at the concentrations that were shown to completely suppress vascular sprouting in a co‐culture angiogenesis assay. We used Ki67 immunostaining to examine if DOX suppresses proliferation of HCMVEC in a co‐culture angiogenesis assay. Endothelial cell marker CD31 was used to identify HCMVEC. DOX markedly reduced numbers of CD31+ and CD31+Ki67+ cells in co‐cultures at concentrations above 4 nM. We conclude that HCMVEC are exceedingly sensitive to anti‐proliferative effect of DOX, and may present a viable target of this cardiotoxic drug in the heart. Endothelial effects of DOX are likely to contribute to the development of dilated cardiomyopathy. This work was supported by NIH NCRR grant 2P20RR016467‐09A1.
Role of p47phox and Nox2 in the Promotion and Impairment of Collateral Growth
Authors: Distasi Matthew , Ingram David , Miller Steven , Unthank Joseph
Published: June 2021

Anti‐oxidant therapy implicates a role for reactive oxygen species (ROS) in both successful collateral growth (CG) and its impairment in models of chronically elevated oxidative stress, including obese mice. NAD(P)H oxidase (Nox) is a major source of vascular ROS. This study investigates the roles of the Nox subunits p47phox and Nox2 in CG (assessed as % increase in collateral diameter compared to same animal control artery) in lean and obese mice. C57BL/6, p47phox−/−, and Nox2−/− mice were fed either standard chow (Lean) or a high‐fat (60% kcal from lard) diet to induce obesity. In lean mice after femoral artery ligation, significant CG occurred in wild‐type (WT) and Nox2−/− mice (171±43 and 112±21%, respectively, p<0.05) but not in p47phox−/− mice (52±31%). In obese mice, CG was greatly suppressed in WT mice (25±13%); however, whole body ablation of both Nox2 and p47phox attenuated the impairment (144±68 and 107±7% increase in collateral diameter, respectively, P<0.05). RT‐PCR analysis demonstrated an increase in p47phox (1.9X, P<0.02) and Nox2 (1.6X, P<0.07) subunit expression in obese vs. lean arteries. Transplantation of WT bone marrow into lean p47phox−/− did not prevent the suppression of CG. In conclusion, these data suggest that tissue resident p47phox is required for CG in lean mice and that obesity‐induced increases in p47phox and Nox2 expression mediate the impairment of CG in obese mice.

Grant Funding Source: NIH T32 HL007910‐26
Hypoxia‐inducible factor‐2α regulates intestinal inflammation and colon cancer
Authors: Shah Yatrik , Xue Xiang
Published: June 2021

A combination of a disturbed function of the intestinal epithelial barrier and a dysregulation of the mucosal immune system are critical in the pathogenesis of Inflammatory bowel disease (IBD). Moreover, IBD is a major risk factor for colorectal cancer. Significant increases in focal hypoxia and its associated transcription factors have been observed during the pathogenesis of colitis and colon cancer. Regulation of hypoxia‐mediated genes is dependent on the nuclear transcription factor, hypoxia‐inducible factor (HIF)1α and HIF2α. HIF1α has been shown to be critical in maintaining the integrity of intestinal epithelial barrier during the pathogenesis of colitis. However, the role of HIF2α in inflammatory lesions in the intestine and colorectal cancer remains less clear. Here we developed novel mouse models with intestine‐specific gain‐of‐function and loss‐of‐function of HIF2α to investigate the role of HIF2α in experimental colitis and colon tumorigenesis. Activation of HIF2α potentiated inflammation, whereas deletion of HIF2α attenuated inflammation in an acute model of colitis. Moreover, HIF2α was critical in the apoptotic response following tissue injury. Since, chronic inflammation is major risk factor for colon cancer and HIF2α was shown to be critical in intestinal inflammation and apoptosis, studies assessing the role of HIF2α in colon carcinogenesis were also performed. The data demonstrated that activation of HIF2α increases colon tumor multiplicity, incidence, and progression. Together, our data suggest an important role of HIF2α as a molecular link between intestinal inflammation and cancer.
Fetal Endothelial Colony Forming Cells from pregnancies complicated by intrauterine growth restriction have reduced vasculogenic capacity
Authors: Bourque Stephane Louis , Sipos Peter , Sibley Colin , Baker Philip , Davidge Sandra , Crocker Ian
Published: June 2021

Background

Diminished numbers of ECFCs may be a risk factor for cardiovascular disease (CVD). Intrauterine growth restriction (IUGR) is a pregnancy complication associated with neonatal morbidity and mortality and long‐term sequelae, including CVD. We hypothesized that fetal ECFCs from human IUGR pregnancies would be fewer in number and exhibit altered vasculogenic potential.

Methods

Cord blood from uncomplicated and IUGR pregnancies were collected at delivery. ECFCs were counted by flow cytometry using discriminators: 7AAD‐/CD31bright/CD45‐/KDR+/CD34+. Vasculogenic potential was investigated by implanting collagen matrices populated with ECFCs into adult immuno‐deficient mice.

Results

ECFC numbers were 67% lower in IUGR cord blood vs. controls (P<0.001), and exhibited a 71% longer doubling time (P=0.01). Blood vessel formation was reduced in collagen implants populated with IUGR‐derived ECFCs. In vitro, hypoxia (1% O2, 72h) induced‐ECFC senescence, and IUGR cells were more susceptible (P<0.05). Finally, IUGR‐derived ECFCs showed a 61% reduction in MMP‐2 release under hypoxic conditions (P<0.001).

Conclusion

ECFCs from IUGR cord blood have intrinsic functional alterations, resulting in diminished vasculogenic potential and increased susceptibility to hypoxia. Supported by grants from the Canadian Institutes of Health Research and the Wellcome Trust.
The Investigation of LPA4 Functions in Zebrafish
Authors: Lin Yu-nung , Kao Shen-Yung , Lee Hsinyu
Published: June 2021

Lysophosphatidic acid, LPA, is a structurally simple lysophospholipid. Derived from phospholipid by autotaxin catalyzation, LPA acts as an extracellular signaling molecule activating various receptors, LPA1–6. There are pleiotropy characteristics of LPA such as wound healing, cancer progression, cardiovascular function, nervous system regulation, reproduction, platelet activation, hair growth, etc. To clarify the function of different LPA receptors, several knock out animals have been obtained. While autotaxin deficient mouse reveal phenotype as embryonic lethality due to vascular defects, none of the single or double deficient LPA1–3 mouse observed similar phenotypes. Here, we investigate the forth LPA receptor, LPA4, which is structural distinct from LPA1–3 in zebrafish. We conducted microinjection of LPA4 morpholino on one cell stage zebrafish embryo. Several phenotypes were observed in LPA4 knock down zebrafish embryos. During early development, edema around pericardial region was observed, suggesting a vascular leakage or failure of lymphatic vessels to absorb body fluids. Also, a significant of decreasing of heart beats with slowed down of blood flow in some individuals. In addition, slightly decrease of intersegmental vessel numbers reveal abnormality of blood vessel development. We conclude that LPA4 may regulate blood and lymph vessel development in zebrafish.
Shear stress upregulates PLGF protein expression in an endothelial cell/vascular smooth muscle cell coculture system
Authors: Rashdan Nabil A. , Lloyd Pamela G.
Published: June 2021

Arteriogenesis, the process of collateral artery remodelling in response to upstream occlusion, can preserve blood flow and prevent ischemic injury. Arteriogenesis is thought to be driven primarily by shear stress. Interestingly, mRNA for the arteriogenic protein placenta growth factor (PLGF) is increased in collaterals by upstream arterial ligation. Thus, we hypothesized that PLGF expression is regulated by shear stress. To model coronary collateral flow (CCF), we utilized a coculture system. Human coronary artery smooth muscle cells (HCASMC) were grown on the bottom of porous inserts, and human coronary endothelial cells (HCAEC) on the top. The HCAEC layer was then exposed to shear for 2 h in a cone and plate device. Three time‐averaged shear conditions were tested: 0.7 dyn/cm2 mimicking normal CCF (NF); 12.4 dyn/cm2 simulating high CCF post‐stenosis (HF); and no shear (C) (N=5 expts). PLGF protein was significantly higher (~25%) in the HF group than in NF or C starting 8 h post‐shear, and continued to increase (~40%) up to 24 h post‐shear (p<0.05). PLGF mRNA was increased 24 h post‐shear in HCASMC in the HF group, relative to NF and C. Shear did not affect PLGF mRNA in HCAEC. These findings indicate that shear stress modulates PLGF expression in HCASMC. We propose that upregulation of PLGF mRNA by shear in SMC in our model system is endothelium‐dependent and may involve hydrogen peroxide. Funding: NIH R01 HL084494 (PL).
Impaired dynamic cerebral autoregulation in type 2 diabetes patients is associated with elevated oxidative stress
Authors: Deo Shekhar H , Vianna Lauro C , Kim Areum , Zimmerman Matthew C , Fadel Paul J
Published: June 2021

Previous studies have indicated that an elevation in oxidative stress within the rat brain impairs cerebral autoregulation (CA). Patients with type 2 diabetes (T2D) have been shown to exhibit elevated systemic oxidative stress. However, whether this increase in oxidative stress is associated with impairments in CA at rest or during exercise remains unclear. In 12 men, 6 T2D patients and 6 age‐matched controls, middle cerebral artery blood velocity and blood pressure (BP) were measured at rest and during static handgrip performed at 40% MVC. Dynamic CA was assessed using the rate of regulation (RoR) and total reactive oxygen species (ROS) was measured using electron paramagnetic resonance (EPR) spectroscopy. Compared to controls, patients with T2D had a lower RoR at rest that was further reduced during handgrip suggesting an impaired dynamic CA. Furthermore, T2D demonstrated greater baseline total ROS compared to controls (1.1±0.2×107 T2D vs. 0.6±0.1×107 controls, EPR a.u.; P<0.05). Baseline total ROS demonstrated a significant inverse relationship with resting RoR (R2 = 0.49; P<0.05) as well as the percent change in RoR from rest to handgrip (R2 = 0.34; P<0.05) with the latter relationship being strongest in T2D (R2 = 0.77; P<0.05). These preliminary findings suggest that a greater underlying oxidative stress may contribute to impairments in CA in T2D at rest and during exercise.
THE FEATURES OF PSYCHO – EMOTIONAL PARENT'S STATE WITH 1 TYPE DIABETES MELLITUS
Authors: Zhuszhasarova Meruert , Igenbaeva Bakit , Baikanova Raushan
Published: June 2021

It was a traditional way to pay attention to biomedical aspects of disease in science and practical treatment of diabetes mellitus. These aspects of chronic disease influence on patient's life, his psychical and social status. The experimental – psychological method was used for analysis psycho – features of parents who have children with diabetes mellitus: “Beck Depression Inventory” divides in groups: the first group – identified, it depends on duration of illness and insulin therapy. The special questionnaire for parents is based on problems of children's life in society. There is a high alarm level of parents, who have 4 – 7 years children, that is 12 % higher than children's. The parentˈs alarm taking their children to the hospital was subclinical. The results show that the parents who have children with complications and long period of disease, need in emotional support. The group of children and parents who had complex rehabilitation have a good dynamics. There is a big improvement of psycho – emotional parents and children state, who got the pomp insulin therapy. The parents interview with little children showed us the decrease of their fear and worries. It is important to establish a good contact and relationships with a doctor in adaptation period for pomp insulin therapy for 24 hours. The parents get knowledge and have a good practice in taking care of their diabetes mellitus children.
Hydrogen sulfide mitigates diabetic nephropathy through NMDA receptor mediated renal remodeling
Authors: Kundu Sourav , Tyagi Aaron , Coley Denise , Sathnur Pushpakumar , Sen Utpal
Published: June 2021

Diabetic nephropathy is a leading cause of vascular morbidity and mortality. Recently, the involvement of N‐methyl‐D‐aspartate receptor (NMDAR) and hydrogen sulfide (H2S) in diabetes associated complications has been implicated. Our aim in this study was to determine whether H2S mitigates diabetic nephropathy by modulating gap junction and matrix proteins by antagonizing NMDAR. We used a diabetic model (Akita, C57BL/6J‐Ins1Akita), matrix metalloproteinase‐9 knockout (KO) [(MMP‐9−/−)] and double KO of Akita/MMP‐9−/− mice and in vitro cell culture.

Results

Diabetic kidneys showed decreased levels of H2S and its enzymes CBS and CSE in both mRNA and protein level and increased expression of NMDA‐R1, connexin‐40, ‐43 (Cx‐40,‐43) and MMP‐9. Treatment with H2S reversed these effects. In double KO mice, NMDA‐R1 was high but Cx‐40, ‐43 was normal. Additionally, treatment with NMDA‐R1 blocker dizicilpine (MK801) failed to disrupt connexin. Glomerular endothelial cells treated with high glucose indicated dysregulated Cx‐40, ‐43. Inhibition of NMDA‐R1 by MK801, silencing MMP‐9 by siRNA or H2S treatment preserved gap junction proteins. We conclude that in diabetic nephropathy, remodeling is mediated by NMDA‐R1 associated MMP‐9 activation and H2S plays a significant role in mitigating nephropathy.
The influence of extracellular calcium sensing receptor (CaR) on H+‐ATPases activity in mice kidney
Authors: Fernandez Ricardo , Casare Fernando
Published: June 2021

With the exception of the glomerulus, the CaR is highly expressed in all renal tubular segments. The aim of this project was to investigate the interaction between CaR and proton extrusion by the vacuolar H+‐ATPase (H+‐ATPase) and gastric‐like isoform of the H+/K+‐ATPase in mice nephron. Biochemical activity of H+‐ATPases was performed using a partially purified membrane fraction of mice cortex and outer medullar region. The inorganic phosphate (Pi) released in the reaction was quantified by a colorimetric reaction and enzyme activity was expressed in nmol of Pi.min−1.mg−1 of protein. This preparation present a bafilomycin (10−7M) sensitive activity (H+ ATPase), and a Schering 28080 (10−5M) sensitive activity (gastric H+/K+ ATPase), both in cortex and outer medullar region. The H+‐ATPase activity from cortical and outer medullary region was significantly stimulated by increasing the Ca2+o, in a dose‐dependent pattern. In the same way, gastric H+/K+‐ATPase activity was sensitive to changes in Ca2+o levels. A significant increase of H+‐ATPase activity was also observed when the CaR was stimulated with agonists such as 300μM Gd3+ and 200μM neomycin, both in cortex: 9,2 ± 1,2 basal vs 16,5 ± 2,9 with Gd3+, and 12,5 ± 2,2 nmol with neomycin, n=10; and outer medulla: 12,3 ± 1,4 basal vs 28,5 ± 3,3 with Gd3+, and 17,9 ± 2,4 with neomycin (n=10). The cortical and outer medullar gastric H+/K+‐ATPase activity was also stimulated by Gd3+ and neomycin. Finally, cortical H+‐ATPase activity was significantly stimulated by 10−9M angiotensin II (16,6 ± 2,1 basal vs 22,0 ± 2,8 with angiotensin, n=7), and the stimulation of CaR in the presence of angiotensin enhanced significantly these effect: 27,7±2,1 with angiotensin plus Gd3+, n=7, suggesting an interaction in the intracellular signaling pathways involved. These data allow us to conclude that CaR enhance proton secretion in both cortical and outer medulla region of mice kidney, by activation of H+‐ATPase and gastric H+/K+‐ATPase.
The cysteines in NBCe1‐A extracellular loop 3 form intra‐molecular disulfide bonds that determines the transporter surface topography
Authors: Zhu Quansheng , Kao Liyo , Azimov Rustam , Liu Weixin , Newman Debra , Kurtz Ira
Published: June 2021

A common feature among the Na+‐dependent members of the SLC4 bicarbonate transporter family is the presence of four cysteines (Cys) in the glycosylated extracellular loop 3 (EC‐loop 3). We recently determined that EC‐loop 3 is the largest highly exposed surface loop in NBCe1‐A and resides at the dimer interface. In the present study, we extensively analyzed the pattern of disulfide bond formation among the four Cys in the NBCe1‐A EC‐loop 3 and discovered: 1) the four cys in EC‐loop 3 are in a folded conformation and intra‐disulfided; 2) cys 583/585 and cys 630/642 form two disulfide bonds; 3) the first disulfide bond (cys 583/585) determines the number of glycosylation sites in EC‐loop 3; 4) glycosylation of EC‐loop 3 guides the disulfide bond formation between cys 630 and 642; 5) in the absence of the four endogenous cys, NBCe1‐A traffics normally to the plasma membrane, however, unlike wild‐type NBCe1‐A, Asn 582 becomes glycosylated; furthermore, every introduced cys in EC‐loop 3 (Tyr 567 to Asp 647) becomes self cross‐linked. Our findings suggest that the EC‐loop 3 from each NBCe1‐A monomer interact to form a domain‐like structure on the surface of the dimerized transporter whose physiological function is currently unknown.
Role of Purinoceptors in Regulating Afferent Arteriolar Diameter in DOCA‐salt Hypertensive Rats
Authors: Guan Zhengrong , Singletary Sean T , Cook Anthony K , Inscho Edward W
Published: June 2021

Afferent arterioles (AA) are highly responsive to purinoceptor stimulation. ATP contributes importantly to renal autoregulation by activating P2 receptors. Pressure‐mediated, and ATP and β,γ‐methylene ATP (P2X1 receptor agonist)‐induced AA vasoconstriction is impaired in DOCA‐salt hypertensive rats (DOCA rats) while the function of other purinoceptors is unclear. We postulated that P2Y and A1 receptor reactivity is intact in AA from DOCA rats. AA responses to UTP (a P2Y2 receptor agonist) and adenosine were studied in juxtamedullary nephrons. Systolic blood pressure averaged 125±5 and 180±7 mmHg in uninephrectomized (UNx) and DOCA rats, respectively. UTP (10−8 to 10−4 mol/L, n=5) evoked concentration‐dependent vasoconstriction of AA in UNx rats. Diameter declined by 2±1, 6±2, 12±2, 23±5 and 49±9%, respectively. UTP‐induced vasoconstriction was unchanged in DOCA rats (n=5, p>0.05) leading to declines of 3±2, 10±2, 14±3, 29±5 and 43±9%, respectively. The AA response to adenosine was also indistinguishable between two groups (n=6 each, p>0.05). Preglomerular microvascular P2X1 receptor protein expression was unaltered in DOCA rats. These data demonstrate that AA P2Y2 and A1 receptor signaling is preserved in DOCA rats. This study suggests that impairment of AA autoregulatory behavior in DOCA‐salt hypertension cannot be attributed to impaired P2Y2 and A1 receptor signaling.