bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of neutrophil chemotaxis by N‐Acetyl‐Seryl‐Aspartyl‐Lysyl‐Proline and thymosin β4
Authors: Nakagawa Pablo , Xu Jiang , Bordcoch Ginette , Janic Branislava , Carretero Oscar A 
Published: November 2023

The role of neutrophils in heart failure has been extensively analyzed and it has been shown that they are the first cells that massively invade the myocardium after myocardial infarction (MI). N‐Acetyl‐Seryl‐Aspartyl‐Lysyl‐Proline (Ac‐SDKP) is a natural tetrapeptide that is released from its precursor thymosin β4 (TB4). Ac‐SDKP and TB4 are shown to have beneficial effect in post MI cardiac healing by decreasing cardiac rupture and mortality in mice. Therefore we explored whether neutrophils play a role in the mechanism of AcSDKP and TB4 cardiac protection. We hypothesized that Ac‐SDKP and TB4 contribute to heart healing in MI by inhibiting neutrophil chemotaxis.To test neutrophil chemotaxis we performed transwell chemotaxis assay using neutrophil differentiated HL‐60 cells. We measured the migration of a) vehicle, b) Ac‐SDKP (10, 100 nM), or c) TB4 (20 nM) pre‐treated cells towards to a chemotactic bacterial formylated tripeptide N‐formyl‐met‐leu‐phe (fMLP) at 1nM. We also measured the effect of Ac‐SDKP on neutrophil infiltration post‐MI in mice. Ac‐SKDP or vehicle were infused subcutaneously using osmotic minipumps at a dose of 1.6 mg/kg/day. Two days after minipump implantation, MI was induced by permanent ligation of the left descending coronary artery. Neutrophil infiltration was measured at 24 hrs post‐MI by immunohistochemistry.At a dose of 100 nM Ac‐SDKP reduced fMLP‐induced neutrophil chemotaxis by 20 %. However, at 10 nM Ac‐SDKP failed to inhibit neutrophil chemotaxis. TB4 at 20 nM completely inhibited neutrophil migration. In vivo, Ac‐SDKP decreased neutrophil cardiac infiltration (veh: 44 vs Ac‐SDKP: 13 cells/mm2; p < 0.05).We conclude that TB4 is a potent inhibitor of neutrophil chemotaxis, while Ac‐SDKP exerts mild inhibitory effects. Inhibition of neutrophil chemotaxis could explain some of the beneficial effects of Ac‐SDKP and TB4 in post MI cardiac inflammation and fibrosis.Support or Funding InformationSupported by NIH HL028982 and AHA 13PRE17100074
Analysis of phosphorylation of the myosin targeting subunit of smooth muscle myosin light chain phosphatase by Phos‐tag SDS‐PAGE
Authors: Walsh Michael P. , MacDonald Justin A. , Sutherland Cindy 
Published: November 2023

Phosphorylation of the myosin targeting subunit (MYPT1) of myosin light chain phosphatase plays an important role in the regulation of smooth muscle contraction, and several sites of phosphorylation by different protein Ser/Thr kinases have been identified. Furthermore, in some instances, phosphorylation at specific sites affects phosphorylation at neighboring sites, with functional consequences. Characterization of the complex phosphorylation of MYPT1 in tissue samples at rest and in response to contractile and relaxant stimuli is, therefore, challenging. We have exploited Phos‐tag SDS‐PAGE in combination with western blotting using antibodies to MYPT1 to separate multiple phosphorylated MYPT1 species and quantify MYPT1 phosphorylation stoichiometry using purified, full‐length recombinant MYPT1 phosphorylated by Rho‐associated coiled‐coil kinase (ROCK) and cyclic AMP‐dependent protein kinase (PKA). This approach confirmed that phosphorylation of MYPT1 by ROCK occurs at Thr697 and Thr855, PKA phosphorylates these two sites and the neighboring Ser696 and Ser854, and prior phosphorylation at Thr697 and Thr855 by ROCK precludes phosphorylation at Ser696 and Ser854 by PKA. Furthermore, phosphorylation at Thr697 and Thr855 by ROCK exposes two other sites of phosphorylation by PKA. Treatment of Triton‐skinned rat caudal arterial smooth muscle strips with the membrane‐impermeant phosphatase inhibitor microcystin, or of intact tissue with the membrane‐permeant calyculin A, induced slow, sustained contractions that correlated with phosphorylation of MYPT1 at 7 to ≥10 sites. Phos‐tag SDS‐PAGE thus provides a suitable and convenient method for analysis of the complex, multi‐site MYPT1 phosphorylation events involved in the regulation of myosin light chain phosphatase activity and smooth muscle contraction.Support or Funding InformationFunded by the Canadian Institutes of Health Research (MOP‐111262)
Rab25 stimulates functional CaV1.2 channel surface expression in arterial smooth muscle cells
Authors: Bulley Simon , Bannister John P , Leo Marie Dennis , Williams Brent , Jaggar Jonathan H 
Published: November 2023

Plasma membrane‐localized CaV1.2 channels are the primary calcium (Ca2+) influx pathway in arterial smooth muscle cells (myocytes). CaV1.2 channels regulate several cellular functions, including contractility and gene expression, but trafficking pathways that control the surface expression of these proteins are unclear. Similarly, expression and physiological functions of small RabGTPases, proteins that control vesicular trafficking in arterial myocytes are poorly understood. Here, using Western blotting and immunoFRET we show that Rab25, a GTPase previously associated with apical recycling endosomes, is expressed in cerebral artery myocytes. Immunofluorescence resonance energy transfer (immunoFRET) microscopy indicated that Rab25 locates in close spatial proximity to CaV1.2 channels in myocytes. Rab25 knockdown using siRNA reduced surface CaV1.2 abundance in arteries, as determined using arterial surface biotinylation. In contrast, CaV1.2 was not located nearby Rab11A or Rab4 and CaV1.2 surface protein was unaltered by Rab11A or Rab4A knockdown. Rab25 knockdown resulted in CaV1.2 internalization and degradation by a mechanism involving both lysosomal and proteasomal pathways. Rab25 knockdown also reduced whole‐cell CaV1.2 current density in myocytes and depolarization (20–60 mM K+) and pressure‐induced vasoconstriction in cerebral artery segments. These results show that Rab25 is expressed in arterial myocytes and describe a physiological function of this protein. Rab25 promotes CaV1.2 channel surface expression in arterial myocytes to stimulate vasoconstriction.Support or Funding InformationThis work was supported by National Heart, Lung and Blood Institute Grants R01 HL67061, HL094378 and HL110347 to J. H. Jaggar and American Heart Association Scientist Development Grant to M. D. Leo.
Resting tension is important for the contractile response but not to vasodilatation in renal hypertensive rat aorta
Authors: Bendhack Lusiane Maria , Silva Bruno Rodrigues , Grando Marcella Daruge 
Published: November 2023

Nitric oxide (NO) produced by the NO‐Synthase (NOS) controls vascular tone and systemic arterial pressure. Shear stress‐mediated phosphorylation of NOS regulates NOS activity. Phosphorylation of Ser1177 (P‐Ser) activates NOS and Thr495 (P‐Thr) inactivates NOS. Protein kinase Akt/PI3K and c‐Src are enzymes that can phosphorylate these residues, but the signaling in renal hypertensive (2K‐1C) rat aorta submitted to increasing resting tension is not known. This study aimed to evaluate the effects of the resting tension of 1.5 g (control) and 3.0 g on the eNOS phosphorylation in phenylephrine (PE)‐induced contractile response in isolated 2K‐1C rat aorta. Concentration‐effect curves for PE were constructed in aortas under resting tension of 1.5 g and 3.0 g in the absence or presence of NOS inhibitor (L‐NAME, 100 μM). On the top of maintained contraction to PE, concentration‐effect curves for acetylcholine (ACh) were constructed. The expression of P‐Ser and P‐Thr were evaluated by Western Blot. The maximum effect induced by PE on resting tension of 1.5 g was lower in 2K‐1C (1.2 ± 0.2g, n=7; p<0.001) than in normotensive 2K (2.2 ± 0.1g; n=5). This effect was normalized by NOS inhibition with L‐NAME. Both P‐Ser and P‐Thr expressions were higher in 2K‐1C than in 2K aortas. In 2K‐1C the increased P‐Ser was inhibited by PI3K/Akt inhibitor (wortmannin, 20 μM) and the increased P‐Thr was inhibited by c‐Src inhibitor (PP1, 10 μM). Under resting tension of 3.0 g, PE‐induced contractile response was similar in 2K‐1C and 2K aortas. Increased resting tension from 1.5 g to 3.0 decreased P‐Ser and increased P‐Thr in 2K aorta, but it was not modified in 2K‐1C aorta. ACh‐induced relaxation was lower in 2K‐1C (81.4 ± 4.7%; n=7; p<0.001) than in 2K (100.5 ± 2.4%; n=5), that was not modified under 3.0 g resting tension in 2K and 2K‐1C. Taken together, our results demonstrate that the increasing resting tension from 1.5 g to 3.0 g decreases eNOS phosporylation in the activation site (P‐Ser) in 2K rat aorta. The anti‐contractile effect induced by PE in 2K‐1C rat aorta under 1.5 g tension is due to eNOS activation in a PI3K/Akt dependent way. The anti‐contractile effect is abolished by increasing the resting tension to 3.0 g due to P‐Thr in a c‐Src dependent way. These results indicate that resting tension is important for the contractile response, but not to vasodilatation in renal hypertensive (2K‐1C) rat aorta.Support or Funding InformationSupported by FAPESP, CNPq and CAPES.
Circadian rhythms in leukocyte recruitment to tissues
Authors: Scheiermann Christoph 
Published: November 2023

Leukocyte recruitment from blood to tissues is a critical step of the immune system to protect against pathogens and provide tissue repair. While myeloid cell recruitment is a hallmark in inflammatory conditions, lymphocyte recirculation through tissues is an important feature of immune surveillance of an organism at steady‐state. Thus far, various molecules have been implicated in these migratory processes including chemokines and adhesion molecules. However, how this fundamental process of immunity is being regulated at the organismal level at different times is unknown. Here, we describe mechanisms that govern a non‐continuous but oscillatory behavior in the infiltration of myeloid and lymphoid cells to different tissues in the mouse. We screened multiple adhesion molecules and chemokine receptors on various leukocyte subpopulations in addition to molecules expressed by endothelial cells in vascular beds of multiple organs. Our data indicate the existence of a leukocyte‐ and tissue‐specific oscillatory molecular signature in the expression of pro‐migratory factors. Specifically, we present data demonstrating that robust circadian oscillations, exhibiting a period length of ~24h, occur in cellular numbers in murine lymph nodes, showing a ~2‐fold difference with a trough at the onset of the behavioral rest phase (light) and a peak at the onset of the active phase (dark). Oscillations were observed in the capacity of lymphocytes to home to lymph nodes, peaking at the onset of the activity phase. Using lineage‐specific genetic ablation of circadian rhythmicity in T and B cells we demonstrate this to be dependent on both lymphocyte and endothelial oscillations in cell surface receptors. We detected similar oscillations in the capacity of lymphocytes to exit lymph nodes, exhibiting a ~3‐fold difference in the amount of cells in efferent lymphatic vessels. Egress could be modulated in a rhythmic manner acutely by pharmacological interventions as well as chronically with the help of lymphocyte‐specific genetic approaches. Taken together, our data show discrete tissue and leukocyte mechanisms that control rhythmic leukocyte recruitment to organs and provide new insights into the temporal control of innate and adaptive immunity.Support or Funding InformationFunded by the European Research Council (CIRCODE) and the German Research Foundation (Emmy Noether SCHE 1645/2‐1 and SFB914 projects B09 and Z03).
Matrix Metalloproteinase‐12 Reduces Cardiac Dilation Post‐Myocardial Infarction by Decreasing Neutrophil Accumulation
Authors: Iyer Rugmani Padmanabhan , de Castro Brás Lisandra E , Jung Mira , Ma Yonggang , DeLeon‐Pennell Kristine Y , Flynn Elizabeth R , Cannon Presley L , Cates Courtney A , Lindsey Merry L 
Published: November 2023

Early inhibition of matrix metalloproteinase‐12 (MMP‐12) worsens cardiac remodeling post‐myocardial infarction (MI), suggesting a beneficial role for MMP‐12. This study assessed whether exogenous MMP‐12 delivery early post‐MI is an effective therapeutic approach. Male C57BL/6J mice (3–6 months old, n=3/group) were subjected to left coronary artery ligation. Saline or active MMP‐12 (aMMP‐12; 0.5mg/kg/day) infusion was initiated at 3h post‐MI using an osmotic mini‐pump. Mice were sacrificed at day (d) 1 or 5 post‐MI. D0 (no MI) served as a negative control. Infarct areas were similar among saline and aMMP‐12 groups at both d1 and d5 (p=0.76 by ANOVA), indicating similar ischemic injury. Survival rates were not significantly different (p=0.38 at d1, p=0.45 at d5). End diastolic volume (EDV) increased in the saline group, and this increase was attenuated in aMMP‐12 (d0= 45±1 μl, saline= 117±4 μl, aMMP‐12= 85±4 μl). At d1 post‐MI, 12 pro‐inflammatory genes were reduced in the aMMP‐12 group (all p<0.05 vs. saline), including Il8rb, Il‐18, Tgf‐β1, and Tnf. Consistent with cytokine levels, neutrophils were reduced in the aMMP‐12 group by 60% at d1 and by 130% at d5 post‐MI (both p<0.05 vs respective saline groups). Neutrophil removal occurs mainly via apoptosis, and we observed a 4‐fold increase in cleaved caspase‐3 in the aMMP‐12 group at d1 and a 2‐fold increase at d5 post‐MI, compared to respective saline groups (all p<0.05). Our results revealed that MMP‐12 plays a cardioprotective role early post‐MI by attenuating neutrophil infiltration and enhancing neutrophil removal to reduce inflammation and LV dilation.Support or Funding InformationThis work was supported by the American Heart Association 14POST18770012, 14SDG18860050 and 15SDG22930009, the National Institutes of Health 1R03EB009496, SC2HL101430, HHSN 268201000036C (N01‐HV‐00244), HL075360, P01HL051971, P20GM104357, and the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development Award 5I01BX000505
Expression of Bim in Retinal Endothelial Cells or Pericytes is Essential for Normal Regression of the Fetal Vasculature
Authors: Sorenson Christine , Wang Shoujian , Zaitoun Ismail , Johnson Ryan , Jamali Nasim , Sheibani Nader 
Published: November 2023

Apoptosis plays a central role in developmental and pathological vessel regression in the retina. Endothelial cells (EC) and pericytes (PC) each play unique roles during development, maintenance and regression of the retinal vasculature. Bim is a pro‐apoptotic Bcl‐2 family member that plays a prominent role in both normal and pathologic retinal vessel regression. To determine whether retinal vascular regression is attributable to Bim expression in EC or PC we generated mice carrying a conditional Bim allele (BimFlox/Flox) and VE‐cadherin‐cre (BimEC mice) or PDGR‐β‐cre (BimPC). BimEC and BimPC mice demonstrated attenuated hyaloid vessel regression and postnatal retinal vascular remodeling, perhaps as a result of decreased apoptosis. Interestingly, even though conditional Bim mice displayed increased numbers of EC and/or PC their retinal vasculature still was susceptible to hyperoxia‐mediated vessel obliteration and subsequent neovascularization during oxygen‐induced ischemic retinopathy, unlike germline deleted Bim‐deficient mice. Thus, understanding the cell autonomous role Bim plays in the retinal vascular homeostasis will give us new insight into how to prevent pathological retinal vessel regression and preserve vision.Support or Funding InformationRRF/Daniel M. Albert Chair and Lions Pediatric Ophthalmology Research Fund
VEGFR2 Signaling is Decreased During Necrotizing Enterocolitis
Authors: Yan Xiaocai , Liu Xueli , Managlia Elizabeth , De Plaen Isabelle 
Published: November 2023

The pathogenesis of necrotizing enterocolitis (NEC), a common gastrointestinal disease affecting premature infants, remains poorly understood. We previously found that intestinal vascular endothelial growth factor A (VEGF‐A) expression is decreased in human NEC samples and in a neonatal mouse NEC model prior to detectable histological injury. Here we hypothesized that VEGF receptor VEGFR2 is developmentally regulated in the intestine during the perinatal period and that VEGFR2 expression and activation may be altered during NEC development. To test these hypotheses, fetal and neonatal mice of different ages (E16, 18, 20, D0, D1 and D2) were sacrificed. Also, neonatal mice were submitted to a NEC protocol consisting of adult commensal bacteria inoculation/hypoxia/cold stress/formula feeding, or allowed to be dam fed. Pups were sacrificed 24 hours later and their intestine processed for western blot analysis or immunofluorescence analysis of VEGFR2 and several cell‐type specific markers. We found that: 1) VEGFR2 protein expression significantly increases during late fetal life but decreases sharply after birth, remaining low until day of life 2, when it is significantly upregulated; 2) VEGFR2 is only detected in endothelial cells (CD31+ cells) but not in CX3CR1+ myeloid cells; 3) both VEGFR2 and pVEGFR2 are significantly decreased in the intestine of neonatal mice subjected to the NEC protocol. In conclusion, our data suggest that VEGFR2 is developmentally regulated in the perinatal intestine and VEGFR2 expression and activity are decreased during NEC development. We speculate that VEGFR2 signaling may play a critical role during NEC development.Support or Funding InformationNorthwestern Memorial Foundation (Friends of Prentice), NIH grants R01HD060876 (IDP)
Vitamin D Lowers Blood Pressure, ET‐1 and sFlt‐1 in AT1 Autoantibody Induced Hypertension During Pregnancy
Authors: Faulkner Jessica , Amaral Lorena M , Cornelius Denise , Cunningham Mark , Ibrahim Tarek , Thomas D'Andrea , Herse Florian , Dechend Ralf , LaMarca Babbette 
Published: November 2023

Autoantibodies to the angiotensin II type I receptor (AT1‐AA) play a role in hypertension associated with preeclampsia (PE). We found that Vitamin D (VD) reduces AT1‐AA and blood pressure (MAP) in a rat model of PE. Here we tested the hypothesis that VD would improve MAP and endothelial dysfunction markers associated with AT1‐AA during PE. We infused AT1‐AA (1:40 in saline) via osmotic minipump on gestational day (GD) 12–19 in pregnant rats. Either VD2 (270 IU/day) or VD3 (15 IU/day) were given on GD14–18 by oral gavage. Uterine artery resistance index (UARI) was measured by Doppler sonography on GD18 and MAP measured on GD19 by indwelling catheter. MAP increased in AT1‐AA (121±4 mmHg) compared to normal pregnant (NP) (101±2) but was decreased in AT1‐AA+VD2 (105±2) and AT1‐AA+VD3 (109±2). UARI increased in AT1‐AA rats to 0.57±0.01 compared to NP (0.41±0.02). VD3 reduced UARI to 0.49±0.03. Placental preproendothelin‐1 (ET‐1) mRNA expression increased in AT1‐AA (18.1±2.9‐fold) from NP and was decreased with VD2 (4.3±1.4) and VD3 (1.3±0.3). Plasma 8‐isoprostanes (ROS) increased in AT1‐AA to 1633±179 pg/ml compared to NP (634±197) but was decreased with VD2 (436±81) and VD3 (752±94). sFlt‐1 increased in AT1‐AA (715±189 pg/ml) compared to NP (94±12) and was reduced with both VD2 (77±16) and VD3 (195±7). We have demonstrated that VD improved ET‐1, ROS and sFlt‐1 in response to AT1‐AA in pregnant rats. We believe reducing these factors led to reduced vascular resistance and MAP responses commonly seen in elevated AT1‐AA, as in the case of PE, thereby supporting a role for VD supplementation as a prevention for PE.Support or Funding InformationProject funded by NIH RO1HD067541 and T32HL105324
Homo‐ and Hetero‐dimerization of Uteroplacental and Uterine Arterial Matrix Metalloproteinase‐9 during Hypertension‐in‐Pregnancy
Authors: Chen Juanjuan , Ren Zongli , Zhu Minglin , Khalil Raouf A 
Published: November 2023

Preeclampsia is a complication of pregnancy manifested as hypertension‐in‐pregnancy (HTN‐Preg) and often fetal growth restriction. Placental ischemia could be an initiating event, but the linking mechanisms leading to HTN‐Preg and growth restriction are unclear. To test the hypothesis that matrix metalloproteinases (MMPs) play a role in HTN‐Preg, the expression and activity of MMP‐2 and ‐9 and tissue inhibitor of MMP (TIMP‐1) were measured in uterus, placenta and uterine artery of Norm‐Preg rats and rat model of reduced uteroplacental perfusion pressure (RUPP). Consistent with our previous report, blood pressure (BP) was higher and gelatin zymography showed reduction in MMP‐2 (~60kDa), pro‐MMP‐2 (~70kDa), MMP‐9 (~80kDa) and pro‐MMP‐9 (~90kDa) in uterus, placenta and uterine artery of RUPP vs. Norm‐Preg rats. Careful examination of the zymographs showed additional bands at 220kDa, 200kDa and 135kDa. Western blots with MMP‐9 antibody suggested that the 200kDa band may represent MMP‐9 homodimer. Western blots with TIMP‐1 antibody as well as reverse zymography suggested that the 220kDa and 135kDa bands may represent MMP‐9 heterodimers (MMP‐9 homodimer/TIMP‐1 and MMP‐9/TIMP‐1, respectively). Importantly, the amount and gelatinase activity of MMP‐9 homodimer was decreased while MMP‐9 heterodimers were increased in uterus, placenta and uterine artery of RUPP vs. Norm‐Preg rats. Thus the increased BP in placental ischemia model of HTN‐Preg is associated with a decrease in MMP‐9 homodimer and an increase in MMP‐9/TIMP‐1 heterodimers in uterus, placenta and uterine artery, which together with the decrease in MMP‐2 and ‐9 could lead to a net decrease in collagenase activity, increased uteroplacental and uterine arteriolar collagen, and growth‐restrictive remodeling in the uteroplacental circulation in HTN‐Preg.Support or Funding InformationThis work was supported by grants from National Heart, Lung, and Blood Institute (HL‐65998,HL‐111775).
Perinatal taurine depletion depresses the renal tubular effect of estrogen in adult female rats independent of high sugar intake
Authors: Roysommuti Sanya , Lerdweeraphon Wichaporn , Wyss James Michael 
Published: November 2023

Perinatal taurine exposure affects adult renal function. This study tests the hypothesis that the adverse effect of perinatal taurine depletion on renal excretory function in adult female rats is modified by estrogen status. Female Sprague‐Dawley rats were fed normal rat chow and given water alone (C) or water containing 3% beta‐alanine (taurine depletion, TD) from conception until weaning. After weaning, the rats received normal rat chow and tap water with (CG, TDG) or without (CW, TDW) 5 % glucose. At 7–8 weeks of age, renal function at rest and after acute saline load (5% body weight) was tested in conscious, restrained female rats treated with or without estrogen receptor blockade (tamoxifen; 10 mg/kg/day in drinking water for a week). Body, heart, and kidney weights were not significantly different among the eight groups. Compared to control, TD did not affect mean arterial pressure (MAP) or heart rate. Tamoxifen treatment significantly increased only resting MAP in TDG compared to TDW and heart rate at rest and after saline load in CG compared to CW groups. Effective renal blood flow, renal vascular resistance, glomerular filtration rate, and sodium and water excretion were not significantly different among the corresponding (tamoxifen treated and untreated) groups. Fractional sodium and water excretion were markedly and significantly depressed after a saline load in CG, TDW, and TDG compared to CW groups, but these differences were eliminated by tamoxifen treatment. These data indicate that in adult female rats, the adverse effects of perinatal taurine depletion on the renal tubule is modified by estrogen status, independent of high sugar intake.Support or Funding InformationKhon Kaen University