bluuebunny/crossref_metadata_2025 huggingface dataset:
Andrographolide induces the COX‐2 expression in MC3T3 – E1 cells via the ERK1/2 pathway
Authors: Perez Tania , Hidalgo Maria A , Carretta Maria D , Hancke Juan L , Burgos Rafael A
Published: June 2021

Andrographolide is a labdane diterpene that possess antiinflamatory properties. This drug reduces iNOS, IL‐8 and COX‐2 expression in macrophages and neutrophils interfering with NF‐κB binding. COX‐2 are involved in the prostaglandins synthesis during inflammatory process, and selective COX‐2 inhibitors have been useful for rheumatoid arthritis and osteoarthritis treatment; however it is widely recognized that COX‐2 inhibition can also interfere with the bone repair, affecting the fracture healing. In this work we assess if androgrpholide can interfere with the COX‐2 expression induced by TNF‐α (proinflammatory cytokine) or TGF‐β (bone growth factor) in MC3T3 – E1 murine preosteoblastic cells. We analyzed the ERK1/2 and SMAD 2–3 phosphorylation and COX‐2 expression by western blot. By Q‐RT‐PCR we assess the effect of andrographolide on COX‐2, cbfa1 and NF‐κB mRNA level in cells treated with TNF‐α or TGF‐β. We demonstrated that andrographolide potentiates the COX‐2 expression and ERK1/2 phosphorylation induced by TNF‐α or TGF‐β in MC3T3 – E1 cells. Moreover we observed that andrographolide by itself was able to stimulate the ERK1/2 phosphorylation and COX‐2 expression in a dose dependant manner. We conclude that andrographolide can modulate directly the osteoblast activity inducing COX‐2 expression and potentiating other stimuli via ERK1/2 pathway.

This work was supported by FONDEF D04I1240
Adeno‐Sh Beta Catenin Abolishes Ischemic Preconditioning Mediated Cardioprotection by Downregulation of its Target Genes VEGF, Bcl‐2 & Survivin In Rat Myocardial Ischemia Model
Authors: Thirunavukkarasu Mahesh , Zhan Lijun , Han Zhihua , Menon Venugopal P , Maulik Nilanjana
Published: June 2021

β‐catenin (β‐cat) is a downstream target of Glycogen Synthase Kinase‐3β (GSK‐3β) that plays a vital role in ischemic preconditioning (IP) mediated angiogenesis & cardioprotection. In the present study we investigated the mechanism of IP mediated cardioprotection through cytosolic β‐cat stabilization, nuclear translocation & activation of its target genes VEGF, Bcl‐2 & survivin. β‐cat expression was suppressed by intramyocardial injection of adenovirus associated sh‐RNA against β‐cat (Ad‐sh‐β‐cat, 4×108 pfu). Adeno‐LacZ was used as the control. The rats were assigned to four groups (1) Control‐IR (CIR) (2) Ad‐LacZ + IR (LIR) (3) Ad‐LacZ + IP (LIP) (4) Ad‐sh‐β‐cat + IP (sh‐β‐catIP). 72h after injection, isolated hearts were subjected to 30min of ischemia [I] & 2h of reperfusion [R] (IR). Designated hearts were subjected to IP (5min of I + 10min R, 4 cycles) before IR. Significant reduction in left ventricular functional recovery throughout reperfusion & increased infarct size were observed in sh‐β‐catIP compared to LIP. Real time PCR & RT‐PCR analysis showed significant downregulation of mRNA expression of VEGF, Bcl‐2 & survivin in sh‐β‐catIP compared to LIP. These findings indicated for the first time that silencing β‐cat abolished IP mediated cardioprotection, thereby validating that β‐cat activation & nuclear translocation is necessary for the IP mediated expression of VEGF, Bcl‐2 & survivin.
Gel electrophoresis and multiplexed fluorescent imaging analysis for the measurement of protein thiol oxidation induced in murine airway epithelial cells after treatment with the Clara cell toxicant, naphthalene (NA) or the non cytotoxic thiol depletor, diethylmaleate (DEM)
Authors: Spiess Page Caroline , Morin Dexter , Buckpitt Alan
Published: June 2021

Oxidative stress leading to alterations in airway cell protein thiol (PSH) status has been implicated in NA cytotoxicity. This study identified specific thiol containing proteins altered by NA or DEM. Murine airway epithelial PSH and formation of oxidized thiols (PSSP, PSSG, PSOH, PSO3) in response to NA (200 mg/kg, ip) or DEM (1 g/kg, ip) was measured using multiplexed fluorescent 2D gel electrophoresis. Proteins were labeled with SH reactive Cy3, disulfides and sulfenic acids were reduced and labeled with Cy5. Proteins were separated to measure significant changes in the SH/oxidized ratios of each spot using the ratio of maleimide labels. NA and DEM caused significant changes in PSH of 82 proteins total compared to control (p<0.05). DEM oxidized more proteins to a greater extent than those recovered from murine airway epithelial samples after NA. However, several proteins containing reactive thiols are oxidized to a greater extent by NA compared to DEM including those involved in transcription regulation (zinc finger proteins), protein synthesis/folding (ribosomal proteins, serine peptidase inhibitor), cell growth/cycle control, and apoptosis (Suv39h2 Histone‐lysine N‐methyltransferase, Bcl2‐associated transcription factor 1). We conclude that specific proteins/protein pathways are disrupted by oxidation/arylation in response to NA that are less affected by nontoxic doses of DEM. NIEHS 04311/04699
γ‐H2AX Formation is not Coupled to Elevations in Intracellular Ca2+, and Hyperactivation of PARP does not Facilitate AIF Translocation During ROS‐induced Cell Death.
Authors: XIE Ruiyu , Lau Serrine S , Monks Terrence J
Published: June 2021

2,3,5‐tris(Glutathion‐S‐yl)hydroquinone (TGHQ) is a redox‐active metabolite of hydroquinone, and causes non‐apoptotic cell death in renal proximal tubule epithelial cells (HK‐2) via the generation of reactive oxygen species (ROS). TGHQ induced ROS generation and DNA strand breaks leads to the overactivation of poly(ADP‐ribose) polymerase‐1 (PARP‐1), the rapid depletion of nicotinamide adenine dinucleotide (NAD+), and the appearance of γ‐H2AX foci. Inhibition of PARP‐1 completely blocked TGHQ‐mediated poly(ADP‐ribosyl)ation (PAR), NAD+ consumption, and the consequent necrotic cell death. Moreover, although Ca2+ chelation inhibited PAR and significantly abrogated cell death, NAD+ depletion was only delayed and not prevented. Despite their ability to inhibit cell death, neither PARP inhibition nor Ca2+ chelation altered ROS production or the initial DNA damage (γ‐H2AX foci). Interestingly, PARP‐1 hyperactivation was not accompanied by the translocation of apoptosis‐inducing factor (AIF) from mitochondria to the nucleus, a process usually associated with PARP‐dependent cell death. In conclusion, PARP activation and Ca2+ elevation are critical responses to ROS‐mediated cell death, but at least in HK‐2 cells γ‐H2AX formation is not coupled to elevations in intracellular Ca2+, and PARP activation does not facilitate AIF translocation. (DK59491, P30 ES 06694).
Decreased microvascular functional vasodilatory reserve and features of the metabolic syndrome
Authors: Clough Geraldine F , L’Esperance Veline , Turzyniecka Magdalena , Krentz Andrew , Byrne Christopher D
Published: June 2021

We have investigated insulin‐induced changes in microvascular conductance in individuals with features of the metabolic syndrome using laser Doppler fluximetry (LDF) and a novel 785 nm, 20 mW, 4 mm separation probe (Moor Instruments Ltd, UK). 33 volunteers [52±9 y (mean ±SD); 15 men)] were studied. All gave written informed consent. LDF was measured above the anterior tibialis muscle of the non‐dominant lower limb and insulin sensitivity by hyperinsulinaemic euglycaemic clamp. Measurements were made at rest and during reactive hyperaemia (RH) to arterial occlusion, before and during the clamp. Vascular conductance (VC) calculated as LDF/MABP (arbitrary units), at baseline and during RH, decreased with >3 features of the metabolic syndrome (p=0.01) and calculated overall CVD risk (p=0.01). VC at was significantly increased by insulin at baseline (0.8±0.4 vs 1.1±0.6, p=0.001) but reduced during peak RH (before insulin: 413±32 vs high dose insulin: 363±35 %baseline, p=0.01). The impaired RH response (peak VC and rate of recovery from peak) was associated with plasma total cholesterol (r2=0.51, p=0.01) and LDL cholesterol concentrations (r2=0.49, p=0.05). We conclude that individuals with features of the metabolic syndrome show a reduced functional vasodilatory reserve and that this impaired response contributes to a reduced insulin sensitivity and glucose handling by muscle.
Angiotensin receptor blocker (ARB) treatment causes paradoxical microvascular endothelial cell proliferation
Authors: Munzenmaier Diane H. , Greene Andrew S.
Published: June 2021

Aim: Previously our laboratory has shown that cerebral angiogenesis in rats is stimulated by chronic treatment with the angiotensin II (ANG II) type 1 receptor (AT1R) antagonist, Losartan (LOS). Because the pro‐angiogenic actions of ANG II via AT1R have been well documented in the peripheral microvasculature, the finding that an AT1R antagonist stimulates cerebral angiogenesis is paradoxical. The aim of this study is to determine whether the in vivo findings are due to receptor blockade per se or whether LOS might exert nonspecific actions in the cerebral vasculature that could explain the in vivo paradox.

Methods: Primary human cerebral and dermal microvascular endothelial cells (ECs), passage 3–8, were seeded into 96‐well plates (5000 cells/well) and allowed to adhere for 24 hrs. Cells were exposed to serum‐free media (SFM) alone and in the presence of LOS (10 μM), ANG II (1 μM) or both for 48 hours. Cell density was measured by a fluorescent cell proliferation assay (CyQUANT, Molecular Probes) on a plate reader at 480/520 nm.

Results: Cell density increased with ANG II treatment of both cerebral (27.5%) and dermal ECs (8.9%) compared to SFM. LOS treatment elicited a similar response (cerebral: 27.1%, dermal: 19.0%). Treatment with both LOS and ANG II, which would simulate AT1R blockade, also stimulated EC proliferation (cerebral 33.7%, dermal: 15.2%).

Conclusion: Since LOS alone, either in the presence or absence of agonist, stimulated cell proliferation in cells derived from both brain and skin, it would suggest that 1) LOS stimulates EC proliferation directly, not by receptor blockade, and 2) tissue differences are not detectable in cultured ECs derived from neural tissue compared to peripheral tissue. Future studies are needed to determine the mechanism for this effect and the cause of observed tissue differences in vivo.

Supported by NIH HL29587 and HV28182
Using micro‐PIV to interrogate the endothelial surface layer in arterioles in vivo
Authors: Savery Michele D , Damiano Edward R
Published: June 2021

The existence of a hydrodynamically relevant endothelial surface layer (ESL), ~ 0.5 μm in thickness, is well established in capillaries and venules in vivo. Since the ESL on vascular endothelial cells is likely to have broad implications for endothelial‐cell function, including mechanotransduction and vascular permeability, it is necessary to determine the extent to which the ESL is present on arteriolar endothelium. We applied microviscometric analysis to data obtained using micro‐particle image velocimetry (μ‐PIV) in cremaster‐muscle arterioles of wild‐type mice. Due to the puslatile nature of the flow in arterioles, data acquisition was triggered with the EKG at specific points in the cardiac cycle. Results from data taken at peak systole in the cardiac cycle show the existence of a hydrodynamically relevant ESL that is ~ 0.38 μm in thickness in arterioles 20–60 μm in diameter (n=18). This is compared with results from venules showing a hydrodynamically relevant ESL that is ~ 0.53 μm in thickness (n=23). We therefore conclude that there exists a significant ESL in arterioles that is thinner then that found in venules (p=0.055). Results from data obtained at four equally spaced points in the cardiac cycle show that the ESL thickness in arterioles does not fluctuate with flow variations arising from cardiac‐induced pulsatility (n=8). Supported by NIH grant R01 HL76499.
Arteriolar dilation in response to skeletal muscle contraction is reduced in Cx40‐deficient mice
Authors: Milkau Malte , de Wit Cor
Published: June 2021

Active hyperemia requires substantial dilation in down‐ and upstream vessels. The ascending dilation may involve gap junctions (GJ) which allow the spread of a dilation into upstream vessels. GJ are composed of connexins (Cx) and of these Cx40 is crucial to support a conducted dilation initiated by endothelial agonists. We studied whether Cx40‐dependent communication contributes to arteriolar dilations during active hyperemia in the cremaster microcirculation in vivo using wildtype (wt) and Cx40‐deficient mice (Cx40ko).

The nerve supplying the cremaster muscle was stimulated for 1 to 60 s at 10 or 100 Hz intermittently (burst 0.5, break 2.5 s). Arterioles were monitored by intravital microscopy and diameters measured off‐line. Responses were studied before and after L‐nitro‐arginine and indomethacin (LN+indo).

The amplitude and the duration of the arteriolar dilation was graded with stimulation time and frequency. At 100 Hz the dilation amounted to 24% (1s), 56% (15s), and 83% of the maximal dilation (60s) in wt. 10 Hz stimulation produced about half of these responses. After LN+indo the dilation was only reduced after maximal stimulation (60s 100 Hz: 63%, p<0.05). In Cx40ko mice, the dilation was significantly reduced (15s: 32%; 60s: 58%) compared to wt. In mice carrying a replacement of Cx40 by Cx45 dilations were also reduced but to a lesser degree.

We conclude that Cx40‐dependent communication contributes to arteriolar dilations in response to exercise. This function of Cx40 can only be partially replaced by Cx45. This suggests that the endothelium provides a Cx40‐dependent conduction pathway whose function during exercise cannot be compensated by other dilator mechanisms.
Pannexin protein expression in the rat middle cerebral artery
Authors: Chen Jie , Burns Alan R. , Phillips Sharon C. , Sokoya Elke M.
Published: June 2021

Pannexins are a recently discovered family of proteins that could potentially be involved in cell‐cell communication. The expression pattern of pannexin 2 (Panx2) is thought to be predominantly confined to neuronal cell populations. In the present study, we report for the first time that pannexins are expressed in the cerebrovasculature. By using a combination of RT‐PCR, immunoblotting, immunohistochemistry and immunogold electron microscopy techniques, we characterized the expression pattern of pannexins in the rat middle cerebral artery (MCA). We report that pannexin 1 (Panx1) is weakly expressed in smooth muscle but not in endothelium, and that Panx2 is expressed in both endothelium and smooth muscle. The identification of pannexins in the rat MCA, indicates that pannexin expression is not limited to neuronal cell populations within the central nervous system. Ultrastructural studies using immunogold electron microscopy revealed that Panx2 is associated with both junctional (gap junctions) and nonjunctional areas of the plasma membrane. These data, suggest that Panx2 may play a role in supporting both cell‐cell coupling and hemichannel activity within the cerebrovasculature.

Supported by NIH HL72954, EY017120, and HL070537.
Down‐regulation of BMP‐4 Expression in Coronary Arterial Endothelial Cells: Role of Shear Stress and the cAMP/PKA Pathway
Authors: Csiszar Anna , Labinskyy Nazar , Smith Kira E , Rivera Aracelie , Bakker Erik N.T.P. , Jo Hanjoong , Gardner Jason , Ungvari Zoltan
Published: June 2021

Bone morphogenetic protein 4 (BMP‐4) is a TGFâ family member cytokine that exerts pro‐inflammatory effects on the endothelium. Atheroprotective levels of shear stress (SS) were shown to control endothelial BMP‐4 expression, however, the underlying mechanisms remained unknown. We found that SS down‐regulated expression BMP‐4 (but not BMP‐2, a related cytokine) in coronary arterial endothelial cells (CAECs) as well as in cultured mesenteric arterioles. In HCAECs 8‐Br‐cAMP, the adenylate cyclase activator forskolin or a PKA activator effectively decreased BMP‐4 expression, mimicking the effects of SS. Indeed, SS induced the nuclear translocation of PKA‐c, and inhibition of PKA attenuated the effects of SS and forskolin on BMP‐4 expression. RNA decay assay and BMP‐4 promoter‐driven luciferase reporter gene assay showed that cAMP regulates BMP‐4 expression at the transcriptional level. Thus, laminar SS and cAMP/PKA pathway are important negative regulators of BMP‐4 expression in ECs. Because BMP‐4 elicits endothelial activation and dysfunction, hypertension and vascular calcification, inhibition of BMP‐4 expression by SS and the cAMP/PKA pathway is likely to exert vasculoprotective effects.
Computational modeling of bimodal resting membrane potential (RP) of arteriolar cells
Authors: Jiang Zhi‐Gen , Chen Fang‐Yi , Ma Ke‐Tao
Published: June 2021

The mechanism of bimodal RP in arterioles is not fully understood. We modeled this phenomenon by three steps. First, we mathematically modeled the whole‐cell I/V with a sum of five currents: (1) IKir=(Vm−EK)*GMAX‐Kir/(1+exp((Vm−V0.5Kir)/kKir)); (2) IKdr=(Vm−EK)*(GMAX‐Kdr − GMAX‐Kdr/(1+exp((x−V0.5Kdr)/kKdr))); (3) IKnon‐v=(Vm−EK)*GKnon‐v; (4) Icat= (Vm−ECat)*Gcat and (5) Ipump=(Vm−Vr‐pump)*Gpump. Using the sum of these equations and initial parameters estimated by vascular experiments, our composed program (in MATLAB) could fit to a ramp‐constructed I/V curve and yield parameter measurements of the individual currents of the recorded cell. Second, using GHK zero‐current equation: Vm (RP) = (EK*GKir + EK*GKdr + EKGKnon‐v + ECat*GCat + Epump*Gpump)/(GKir + GKdr + GKnon‐v+ GCat + Gpump) and the above five equations with approximate parameters, the program's iteration process could search the stable Vm, i.e. the RP, after inputting an initial Vm value to equations (1) and (2). When we applied normal distribution random values (n≥50000) of the yielded GmaxKir and one or more of other conductances, the program produced a bimodal distribution of RPs peaked at ∼−75 and ∼−40 mV. Third, using the program and data yielded, the program simulated the observed RP shifts such as those induced by Ba2+‐inhibition of Kir and NO‐activation of KATP. We conclude that the bimodal RP results from sum between optimal Kir and other membrane currents: generally the other currents set a ∼−40 mV RP background, and the Kir causes a high RP ∼−75 mV only when the background enables a Kir activation that triggers the Kir a positive‐feedback activation. Supported by NIH DC004716 & P30 CD005983
Therapeutic Arteriogenesis via the Ultrasound‐Microbubble‐Targeted Delivery of Fibroblast Growth Factor‐2 (FGF‐2) Bearing Poly(Lactic‐Co‐Glycolic Acid) Nanoparticles
Authors: Chappell John C , Song Ji , Klibanov Alexander L , Price Richard J
Published: June 2021

We are developing a therapeutic arteriogenesis strategy in which ultrasound (US) is used to activate intravenously injected contrast agent microbubbles (MBs) and facilitate the targeted delivery of co‐administered fibroblast growth factor‐2 (FGF‐2)‐bearing poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles (NPs) to skeletal muscle. Two weeks after treatment in a mouse hindlimb ischemia model, gracilis adductor muscles (GMs) exhibited an 18% increase in arteriolar length density when compared to controls [bovine serum albumin (BSA) bearing NPs]. In addition, the maximum diameter of the posterior GM feed artery, a major collateral pathway, was significantly increased with treatment (FGF‐2=156.8±16.4 μm vs. BSA=100.8±8.7 μm), as were the maximum diameters of the transverse arteriolar trees (FGF‐2=52.9±2.4 μm vs. BSA=39.8±1.9 μm). We conclude that FGF‐2‐bearing PLGA NPs stimulate both new arteriole formation and the structural enlargement of existing arterioles following targeted delivery with US and MBs. These results support the use of US in conjunction with MB contrast agents as a platform for the minimally‐invasive delivery of pro‐arteriogenic nanomedicine. Supported by NIH HL74082 and AHA0555511U.
The Requirement of CC‐Chemokine Receptor‐2 (CCR2) Expression by Bone Marrow‐Derived Cells (BMCs) for Arteriogenesis is Stimulus Dependent
Authors: Nickerson Meghan M , Song Ji , Owens Gary K , Skalak Thomas C , Price Richard J
Published: June 2021

The MCP‐1/CCR2‐mediated recruitment of BMCs is necessary for hindlimb arteriogenesis in Balb/C mice, but not in C57BL/6 mice. Here, we tested whether a stronger inflammatory stimulus is needed to trigger CCR2‐dependent arteriogenesis in the C57BL/6 strain. We implanted dorsal skinfold window chambers (WCs) or created hindlimb ischemia in C57BL/6 wild‐type (WT) mice engrafted with either CCR2−/− BMCs (CCR2−/−>WT) or WT BMCs (WT>WT). In WT mice, 1 day after stimulation, MCP‐1 levels rose from “undetectable” to 463 pg/mg in WC tissue but only 17.0 pg/mg in ischemic hindlimb. WC implantation elicited a 66% (28 μm) increase in maximum arteriolar diameter over 7 days in WT>WT mice; however, CCR2−/−>WT mice exhibited no arteriogenesis (−0.5%; −2μm). CD11b staining confirmed diminished monocyte recruitment in CCR2−/−>WT WCs. Hindlimb ischemia created strong arteriogenesis responses in both CCR2−/−>WT and WT>WT mice, confirming that CCR2 expression by BMCs is not required for arteriogenesis in this model. We conclude that the requirement for CCR2 expression by BMCs in arteriogenesis is stimulus‐dependent and correlated to the degree of tissue inflammation. Supported by NIH HL65958 and NIH HL74082.
Femoral artery‐vein shunt steals collateral flow and promotes collateral arteriogenesis
Authors: Yang Hsiao Tung , Bowles Douglas K , Terjung Ronald L , Laughlin Harold M
Published: June 2021

To study high flow‐stimulated collateral artery enlargement, a side to side femoral artery‐vein anastomosis was established in Yucatan pigs (~40 kg, n=7, limited to cage activity). The femoral artery was occluded proximally to the a‐v shunt by an Ameroid occluder placed between the a‐v shunt and a proximal transonic flow probe. Femoral artery blood flows (BF) were monitored daily to evaluate the magnitude and time‐course of BF change. Before femoral artery occlusion, BF through the proximal flow probe increased from ~400 to 700 ml/min, while BF through the distal flow probe (distal to the shunt) were ~100 to 200 ml/min. Following femoral artery occlusion (~ 2 wks post surgery), proximal BF reduced to only ~50 ml/min, while distal BF (i.e., arising from collateral dependent BF delivered distal to the shunt) gradually increased from ~200 to 400 ml/min by 4 wks post‐surgery. Thus, a significant­ly greater collateral‐depen­dent BF was shunted back to the central circulation over time. This placed increasing flow demands on the collateral circuit. Angiograms showed extensive collateral vessel development in the upper thigh area in the leg with the a‐v shunt, primarily arising from the profunda artery. We attribute this collateral vessel enlarge­ment to the apparent high shear stress induced in these vessels that would occur to support flow through the low‐resistance shunt created by the by‐pass. Thus, we conclude that an exceptional enlargement of the collateral circuit can be induced over a rather brief period of time, if the stimulus for collateral development is sufficient.

Supported by NIH grant P01HL 52490‐12
Functional consequences of the collagen/elastin switch in vascular remodeling in Hyperhomocysteinemia
Authors: Steed Mesia Moore , Rodriquez Walter , Tyagi Neetu , Tyagi Suresh C.
Published: June 2021

Homocysteine (Hcy), the cholesterol of the 21st century, has emerged as a novel independent risk factor for the development of cardiovascular disease. Elevated circulating Hcy or hyperhomocysteinemia (HHcy) mediates the atherogenic collagen/elastin switch in vascular tissue and resulting reduction in vascular compliance. We hypothesize that the differential production of NO contributes to oxidative stress and increased oxidative/nitrative activation of matrix metalloprotineases (MMPs) resulting in vascular remodeling in response to HHcy. The overall goal is to elucidate the contribution of the nitric oxide synthases (NOS isoforms); endothelial, neural and inducible in the collagen/elastin switch. Experiments were performed on six groups of animals (WT, eNOS−/−, iNOS−/− with and without Hcy treatment [.67g/L] for 8–12weeks). In vivo echograph was performed to assess aortic timed flow velocity for indirect compliance measurement. Histological determination of collagen and elastin with Trichrome and Van Geison's stains, respectively was performed. In situ measurement of superoxide generation using dihydroethidium was used. Differential expression of eNOS, iNOS, nitrotyrosine, MMP ‐2 and ‐9 and elastin were measured by quantitative PCR and Western blot analysis. The 2% gelatin zymography was used to assess MMP activity. The increase in superoxided and robust activity of MMP‐9in eNOS−/−, WT+HCY and eNOS−/−+Hcy was accompanied by the gross disorganization and thickening of the extracellular matrix along with extensive collagen deposition and elastin degradation (collagen/elastin switch) resulting in a decrease in aortic timed flow velocity. Results show that increased activity of iNOS is a key contributor to HHcy mediated collagen/elastin switch and resulting decline in aortic compliance.
Catalase inhibition effect on exogenous hydrogen peroxide induced vasoconstriction in diseased human arterioles
Authors: Wittenburg April Lee , Phillips Shane , Gutterman David
Published: June 2021

Hydrogen peroxide (H2O2) has been show to be an endothelial derived factor that derived from the endothelium regulates vascular tone and tissue blood flow. In previous studies, inhibition of the endogenous H2O2 scavenger, catalase, increased the sensitivity of H2O2‐ induced dilation in an endothelium‐dependent manner in human adipose microvessels. Since ROS and their scavengers may be upregulated in disease, the purpose of this study was to determine if catalase modulation of H2O2 dilation is greater in the coronary and peripheral microcirculation of patients with coronary artery disease (CAD).Methods: Arterioles (∼150 μm ID) obtained at surgery from patients with or without CAD (atrial appendage and visceral adipose), were prepared for videomicroscopy. Vasomotor responses to exogenous H2O2 (10–10 to 10–3 M) were evaluated in the presence and absence of the catalase inhibitor 3‐amino‐1,2,4‐triazole (ATZ (4x10–2M)). Results: H2O2 dilated adipose [ED50=7.62x10‐6M, n=7] and coronary arterioles [ED50=8.63x10–6M] from CAD patients. Adipose tissue with CAD were not affected by inhibition of catalase with ATZ [ED50=4.31x10–6M, n=7]. Treatment with ATZ did not enhance dilation to H2O2 in any of the tissues tested. (with CAD [ED50=4.19x10–6M]; without CAD [ED50=1.08x10–5M]). Conclusions: H2O2 signaling is less modulated by endogenous catalase in subjects with than those without CAD.
Effects of hypothyroidism, T4 and DITPA replacement on brain vessel density in adult rats
Authors: Schlenker Evelyn Heymann , Hora Megan J , Gerdes A. Martin
Published: June 2021

Altered microvascular structure and function may affect mood and cognitive function in hypothyroid patients. We hypothesized that adult hypothyroid (H) rats will have significantly lower forebrain blood vessel densities (BVD) than euthyroid (E) rats, and that treatment with DITPA (D)‐ (a thyroid hormone analogue) or T4(T) will normalize BVD's. Twenty male rats were divided into 4 age‐matched groups. The E group received no thyroidectomy and no treatment. The other three groups received thyroidectomies and pellets. The H group received a placebo pellet, the D group received an 80mg DITPA‐containing pellet, and the T group received a 5.2mg T4 slow release pellet for 6 weeks. Prior to sacrifice, body weights (BW) and body temperatures (BT) were measured. Immunohistochemistry on forebrain regions was performed on 20μm sections using a monoclonal anti‐platelet endothelial cell adhesion molecule to visualize blood vessels. Blood vessels were counted in 20 fields per animal to calculate BVD. The E group averaged BW's of 548 ± 54g, while the H group averaged BW of 332 ± 19g (p‐value < 0.001). Relative to the control group the D‐treated group was significantly lighter (p‐value < 0.00393) while the T‐treated group was comparable in BW to the E group. The same trends were seen with BT with the largest difference between the E and H groups (p‐value = 0.002). BVD in the E group was 147 ± 12 blood vessels per mm2, and in H group 69 ± 5 blood vessels per mm2 (P=0.013), but similar among the E, D, and T groups. These results show that hypothyroidism decreases BVD in adult rat forebrain regions. Moreover, DITPA and T4 are efficacious in reversing the effects of hypothyroidism on BVD.
Coordination of chromosome replication and cell cycle progression in Caulobacter
Authors: Shapiro Lucy
Published: June 2021

The Caulobacter cell cycle is driven by sequential regulators, starting with the expression of DnaA in G1 and ending with the expression of the CcrM DNA methyltransferase at the end of replication. DnaA both allows replisome formation and is a transcription factor for over 40 replication genes. The timing of dnaA transcription is dependent on the methylation state of its promoter, which in turn is dependent on restricting the synthesis of CcrM to the end of the cell cycle. Replication initiation starts on a fully methylated chromosome. The dnaA gene, positioned near the replication origin, is only read when its promoter is in the fully methylated state. When the replication fork passes the dnaA gene, the dnaA promoter becomes hemi‐methylated and thus inactivated. This methylation‐based regulation of dnaA transcription modulates the temporal levels of DnaA during the cell cycle, providing a ratchet mechanism to govern sequential regulator expression.

The bacterial cell also uses spatial information to coordinate cell cycle events. In Caulobacter, a novel system couples the placement of the FtsZ cytokinetic ring to the initiation of chromosome replication and the bipolar positioning of the duplicated origin regions. A ParA‐like protein, MipZ, forms a complex with the ParB partitioning protein near the origin of replication and moves with the duplicated origin regions to the cell poles. MipZ directly interferes with FtsZ polymerization, thereby restricting FtsZ ring formation to mid‐cell, the region of lowest MipZ concentration. The cellular positioning of MipZ thus serves the duel function of positioning the FtsZ ring in‐between the two nascent sister nucleoids and delaying formation of the cell division apparatus until chromosome segregation has initiated.
MKP‐1 Deficiency Augments LPS‐Induced NO Production in the Lung
Authors: Girton Mark R , Liu Yusen , Nelin Leif D , Chicoine Louis G
Published: June 2021

Pulmonary vasoreactivity is altered in neonatal pulmonary inflammatory diseases such as bronchopulmonary dysplasia, which can lead to ventilation‐perfusion (V/Q) mismatch. Mitogen‐activated protein kinase phosphatase (MKP‐1) is essential in limiting the pro‐inflammatory response to lipopolysaccharide (LPS). We hypothesized that LPS treatment in Mkp‐1−/− mice would result in altered pulmonary vasomotor tone due to augmented expression of inducible nitric oxide synthase (iNOS). We tested this hypothesis by treating Mkp‐1−/− mice and their wildtype littermates with 5 mg/kg LPS ip at 1 and 12 h prior to lung isolation and perfusion. Exhaled NO (exNO) was collected at the 12 h time point and measured. We found that baseline PVR was not different in lungs isolated from untreated Mkp‐1−/− mice or their wildtype littermates. However, baseline PVR was lower at 1 h in lungs from LPS treated Mkp‐1−/− mice compared to their wildtype littermates. ExNO was greater in isolated lungs from Mkp‐1−/− mice compared to their wildtype littermates. These results demonstrate that deficiency of MKP‐1 results in lower PVR, which is mediated by augmented pulmonary NO production. We speculate that targeting iNOS gene expression early in pro‐inflammatory states will prevent altered pulmonary vasomotor reactivity and improve V/Q matching.
20‐Hydroxyeicosatetraenoic acid protects the pulmonary vasculature from apoptosis
Authors: Bodiga Sreedhar , Dunn Laurel K , Gao Ying , Gruenloh Stephanie K , Buonaccorsi Noelle J , Al‐Saghir Rula , Falck John R , Medhora Meetha M , Jacobs Elizabeth R
Published: June 2021

The arachidonic acid derivative, 20‐hydroxyeicosatetraenoic acid (20‐HETE) dilates small pulmonary arteries (~1–3 mm in diameter). We recently showed that 20‐HETE increases generation of superoxide in bovine pulmonary artery endothelial cells (BPAECs) via activation of NADPH oxidase. Superoxide is known to stimulate growth of endothelial cells and activate protein kinases including ERK, p38MAPK, and Akt. We therefore tested if 20‐HETE promoted survival of BPAEC from apoptotic cell death. 20‐HETE (10 nM – 1 microM) produced a concentration‐dependent increase in numbers of BPAECs after serum deprivation and attenuated increases in caspase 3 activity, indicating protection against apoptosis. This pro‐survival action of 20‐HETE was abolished by pharmacological inhibition of phospho‐inositide 3‐kinase and Akt. Lipopolysaccharide (LPS) induced apoptosis in BPAECs as determined by nuclear fragmentation and increased activity of caspase 3 was effectively inhibited by 20‐HETE (1 microM). Further, 20‐HETE also reduced caspase 3 activity in ex vivo cultures of small pulmonary arteries from mouse and exposed to hypoxia followed by reoxygenation. In summary, 20‐HETE protects the pulmonary vasculature from apoptosis induced my multiple insults such as serum deprivation, LPS and hypoxia/reoxygenation. Funding: NIH/NHLBI 68627 (ERJ), 49294 (ERJ), 69996 (MMM), GM 31278 (JRF).
Diabetes Increases Cerebrovascular Permeability: Relevance to Ischemia/Reperfusion Injury
Authors: Li Weiguo , Elgebaly Mostafa M , El‐Remessy Azza , Kozak Anna , Schreihofer Derek A , Dorrance Anne M , Fagan Susan C , Ergul Adviye
Published: June 2021

Diabetes not only increases the risk of stroke but also is associated with a worse stroke outcome. We have shown that the Goto‐Kakizaki (GK) rat model of Type 2 diabetes exhibited smaller infarct size but increased hemorrhagic transformation (HT) following 3 h middle cerebral artery occlusion (MCAO) and 21 h reperfusion. Pial vessels were also more tortuous in this model. Taken together, we hypothesized that there is potential neovascularization and increased permeability in the cerebrovasculature of diabetic rats. The baseline cerebral blood flow (CBF) was assessed by a scanning laser Doppler. The cerebrovascular density and permeability was assessed by fluorescein isothiocyanate labeled dextran and BSA, respectively, in diabetic and control rats without MCAO. Blood glucose was higher (168 ± 5 vs. 108 ± 3 mg/dl, p<0.05) and baseline CBF was lower in the GK rats (2.74 ± 0.20 vs. 3.93 ± 0.16 pixel/mm2, p<0.05). Permeability of cerebral vessels was increased in the GK group (84 ± 9 vs. 47 ± 6 OD, p=0.018). A slightly higher vascular density in GKs (n=3, p=0.18) may be a potential mechanism contributing to smaller infarcts. These findings suggest that diabetes promotes cerebral vascular permeability and accordingly augments ischemia/reperfusion injury induced HT.
Role of Oxidative Stress and Angiotensin II in Cerebral Vascular Dysfunction with Aging
Authors: Modrick Mary L. , Didion Sean P. , Sigmund Curt D. , Faraci Frank M.
Published: June 2021

Endothelial dysfunction occurs with aging. We hypothesized that oxidative stress and angiotensin II (Ang II, via AT1a receptors) promote cerebral vascular dysfunction with aging. We studied young (∼6 mo), old (∼17 mo) and very old (22–25 mo) mice. In basilar arteries, acetylcholine (an endothelium‐dependent agonist) produced dilation in young mice that was reduced by ∼60 and 90% (P<0.05) in old and very old wild‐type mice, respectively. Similar changes were seen in arteries from mice deficient in Mn‐superoxide dismutase (MnSOD +/−). Relaxation of aorta and carotid artery to acetylcholine was normal in old mice and only modestly affected in very old mice. The cerebral vascular ressponse to acetylcholine in very old mice was largely (∼80%) restored with Tempol (a scavenger of superoxide) and partly (∼40%) restored by PJ34, an inhibitor of PARP. Cerebral vascular responses were similar in young mice lacking AT1a receptors and wild‐type mice. Responses of basilar arteries to acetylcholine were reduced by >50% in old AT1a wild‐type (P<0.05). In contrast, vasodilation to acetylcholine was normal in old AT1a deficient mice. Thus, age‐related endothelial dysfunction occurred earlier and to a greater extent in basilar arteries compared to non‐cerebral blood vessels, but was not enhanced by MnSOD deficiency. Our findings suggest a novel role for Ang II in age‐related cerebral vascular dysfunction.
Obesity alters cerebral vascular structure: Correlation with age
Authors: Osmond Jessica Martin , Dalton Brian , Mintz James D. , Stepp David W.
Published: June 2021

Obesity and insulin resistance (IR) have been shown to alter cerebral vascular reactivity and to worsen the outcome of stroke. Vessel structure is one of the factors affecting the outcome of stroke. This study hypothesized that adult obese Zucker rats (OZR), a model of obesity, IR and mild hypertension, would demonstrate inward remodeling of the middle cerebral artery (MCA) indicated by smaller lumen and outer diameters compared to lean Zucker rats (LZR). Passive lumen and outer diameters were measured over a range of intralumenal pressures (0–140 mmHg) in MCAs from adult (13–18 weeks) LZR and OZR. OZR had smaller lumen (222 ± 9 vs. 280 ± 6 μm at 140 mmHg) and outer (255 ± 11 vs. 318 ± 7 μm at 140 mmHg) diameters over the entire pressure range compared to LZR with no difference in wall/lumen ratios. Paradoxically, OZR had a rightward shift in the stress‐strain curve and a smaller P‐coefficient (3.79 ± 0.58 vs. 5.86 ± 0.55 in LZR, p<0.05), indicating increased compliance. We then compared MCAs from young (6–7 weeks) OZR and LZR before the development of hypertension and found no differences in vessel structure or compliance despite increased body weight of OZR and IR. Our results provide evidence of structural changes in the cerebral vasculature of adult OZR. These changes appear to correlate with the development of additional metabolic complications and increased blood pressure. (HL076533–03)
Model prediction of subendocardial perfusion in the presence of an epicardial coronary artery stenosis
Authors: van den Wijngaard Jeroen , Kolyva Christina , Siebes Maria , Dankelman Jenny , van Gemert Martin JC , Piek Jan J , Spaan Jos AE
Published: June 2021

Objective: With coronary disease the subendocardium is most vulnerable to ischemia which will occur when Diastolic Time Fraction (DTF) is to short and/or coronary pressure (Pc) to low. Since recent clinical techniques permit measurement of subendocardial perfusion our objective is to predict the effects of hemodynamic conditions on the induction of ischemia.

Methods: Using data from microsphere experiments a layered model of the myocardial wall was developed in which each layer is represented by a hyperemic resistance and autoregulatory resistance in series. Myocardial perfusion distribution during hyperemia was predicted for different degrees of coronary stenosis, at different levels of DTF.

Results: A reference DTF was chosen (corresponding to HR=90) at which perfusion was rather evenly distributed at normal Pc = 90 mmHg. At this reference condition the effect of the stenosis on perfusion distribution was homogenous. However, at shorter or longer DTF, the subendocardium was the first or last to suffer from shortage of perfusion, the maginitude of which depended on stenosis degree.

Conclusions: For larger values of DTF as normal for patients with medication and with the applied pharmacological stress tests the possible occurrence of subendocardial ischemia at physical exercise is underestimated.
Increased cholesterol is vital to the development of coronary artery disease and type 2 diabetes in Ossabaw swine
Authors: Neeb Zachary Paul , Edwards Jason M , Bratz Ian N , Allosh Mouhamad A , Sturek Michael
Published: June 2021

Ossabaw swine fed excess kcal high fat/cholesterol diet develop metabolic syndrome. Many studies have linked a high‐fructose diet to development of metabolic syndrome without dyslipidemia. We tested the hypothesis that a high fructose diet will induce metabolic syndrome (MetS) and a high fructose/fat/cholesterol diet will induce more severe, dyslipidemic MetS (DMetS). We fed healthy, lean Ossabaw swine control chow (N=9), high fructose diet (MetS; N=8), or high fructose/fat/cholesterol diet (DMetS; N=6) for 28 weeks. Compared to lean pigs, both MetS and DMetS developed obesity, glucose intolerance, hypertriglyceridemia, and hypertension. However, increased cholesterol was seen only in DMetS compared to lean, while MetS was not different from lean. Importantly, DMetS developed type 2 diabetes, as evidenced by significantly increased fasting blood glucose and pancreatic beta‐cell failure (lack of both hyperinsulinemic response to intravenous glucose tolerance test and beta‐cell immunohistochemistry). Intravascular ultrasound revealed very minimal CAD in lean and MetS, but CAD in DMetS was occlusive and diffuse. In conclusion, although several risk factors for CAD are present in MetS Ossabaw swine, dyslipidemia is a crucial component in the development of type 2 diabetes and CAD. The data are consistent with a role of lipotoxicity. Support: NIH RR013223, HL062552.
Oxidized lipids, LOX‐1, and diabetic nephropathy
Authors: Dominguez Jesus , Kelly Katherine J.
Published: June 2021

The early stages of nephropathy in obese, diabetic, dyslipidemic (ZS) rats are characterized by tubular lipid accumulation and pervasive inflammation. Our prior work has shown that these events are interrelated. We now tested the hypothesis that proximal tubules from ZS obese diabetic proteinuric rats in vivo, and cultured proximal tubule cells (NRK52E) exposed to oxidized LDL (oxLDL), changed their normally quiescent epithelial phenotype into a proinflammatory phenotype. Urine of obese diabetic rats contained more lipid peroxides, and LOX‐1, a membrane receptor that internalizes oxidized lipids, was mobilized to luminal sites. Levels of the intercellular adhesion molecule‐1 (ICAM‐1) and focal adhesion kinase (FAK), which participate in leukocyte migration and epithelial dedifferentiation, respectively, were also upregulated in tubules of obese proteinuric rats. NRK52E cells exposed to oxLDL showed similar modifications, plus activation of MAPK enzymes and suppression of anti‐inflammatory transcription factor PPAR delta. In addition, oxLDL impaired epithelial barrier function. These alterations were prevented by an anti‐LOX‐1 antibody. The data support the concept that tubular LOX‐1 activation driven by lipid oxidants in the pre‐urine fluid is critical in the inflammatory changes. We suggest that luminal lipid oxidants and abnormal tubular permeability may be partly responsible for the renal tubulo‐interstitial injury of obesity, diabetes and dyslipidemia.
Carotid artery compliance and systemic nitric oxide synthase inhibition in young healthy adults
Authors: Sugawara Jun , Komine Hidehiko , Yoshizawa Mitsuko , Saito Yoko , Nakamura Mariko , Ajisaka Ryuichi , Maeda Seiji , Tanaka Hirofumi
Published: June 2021

Decreased arterial compliance (increased arterial stiffness) is associated with cardiovascular events. Nitric oxide regulates vascular tone, which can influence arterial compliance. We previously investigated the effects of systemic NOS inhibition on arterial compliance under the systemic α‐adrenergic receptor blocking. In the present study, we investigated the effect of systemic NOS inhibition alone on central arterial compliance. Sixteen apparently healthy young adults (26±1 years) underwent intravenous infusions of NG‐monomethyl‐L‐arginine (L‐NMMA) or placebo (saline) on separate days. In the placebo control condition, no significant changes were observed in mean arterial pressure (68±2 vs. 71±1 mmHg), carotid artery compliance (0.10±0.01 vs. 0.10±0.01 mm2/mmHg), and β‐stiffness index (7.3±0.3 vs. 7.1±0.4 AU). After the administration of L‐NMMA, mean arterial pressure increased significantly (70±2 vs. 79±2 mmHg). However, there were no significant changes in carotid artery compliance (0.09±0.01 vs. 0.10±0.01 mm2/mmHg) or carotid β‐stiffness index (7.3±0.3 vs. 6.7±0.4 AU), an index adjusted for distending pressure. Our findings indicate that systemic NO inhibition does not modulate central arterial compliance in young healthy adults.
Distinguishing endogenous and exogenous sulfide by mass spectroscopy
Authors: Leviten Dina , Mulligan JoAnne , Bengtsson Asa , Insko Michael , Mebel Elise , VandenEckart Emily , Vertz Peter , Toombs Christopher , Wintner Edward A , Szabo Csaba , Deckwerth Thomas
Published: June 2021

Hydrogen sulfide exhibits many characteristics of an endogenous biological mediator and is present at low baseline levels in tissues and blood. A large body of evidence suggests that exogenously applied sulfide may also be suitable as a therapeutic agent. Therefore, a method using the rare 34S sulfur isotope was developed and validated to distinguish exogenously administered sulfide from endogenous sulfide which is predominantly 32S‐sulfide. Sodium 34S‐sulfide was prepared and administered intravenously to Sprague Dawley rats. Concentrations of 32S and 34S‐sulfide were measured in blood and tissues after derivatization with monobromobimane as monobromobimane derivative, separation by reverse phase HPLC and quantification by mass spectroscopy. On intravenous infusion of sodium 34S‐sulfide into rats, blood sulfide concentrations of 34S‐sulfide, but not 32S‐sulfide increased in a dose‐dependent manner indicating that exogenously administered sulfide can be traced and distinguished from endogenous sulfide. The method will be useful in monitoring of the distribution of therapeutic sulfide in tissues and organs.
Paradoxical effects of N‐Cadherin overexpression in embryonic stem cells
Authors: Gillum Nikki , Karabekian Zaruhi , Sarvazyan Narine
Published: June 2021

Pluripotent embryonic stem cells (ESC) are a promising source for the regeneration of damaged tissues. To date, it is not known whether genetic modification of ESC can promote their integration into the host tissue and whether upregulation of the adhesion proteins would facilitate this process. We tested a hypothesis that overexpression of a major adhesion protein, N‐Cadherin, will alter the adhesive properties of embryonic stem cells. Constitutive overexpression of a major adhesion protein, N‐Cadherin, in an embryonic stem cell line led to marked changes in the phenotype and adhesion properties of these cells. These changes occur despite the lack of a detectable increase in membrane‐associated N‐Cadherin. The level and the membrane distribution of gap junctional protein Connexin‐43 and the amount of p120‐catenin are elevated in N‐Cadherin overexpressing cells, pointing to altered protein‐protein interactions at cell junctions. The data suggest that while the overexpression of N‐Cadherin makes embryonic stem cells more adhesive, the effect might be paradoxically mediated by other proteins. Financial support by the American Heart Association, MidAtlantic Affiliate (0715335U) and the NIH (HL076722, HL087529) is gratefully acknowledged.