bluuebunny/crossref_metadata_2025 huggingface dataset:
Estrogen‐Dependent Upregulation of the Renin Angiotensin System and Constitutive NOS Accounts for Ethanol Enhancement of Enalapril‐Evoked Hypotension in Telemetered Female Rats
Authors: El‐Mas Mahmoud , Abdel‐Rahman Abdel
Published: June 2021

We have previously reported that chronic ethanol enhances the hypotensive effect of enalapril in female rats. Here, we investigated whether this effect of ethanol is estrogen‐dependent and whether the renin‐angiotensin system (RAS) and nitric oxide synthase (NOS) constitute molecular targets for the ethanol‐enalapril interaction. Four groups of telemetered rats were employed, 2 ovariectomized (OVX), and 2 estrogen‐replaced OVX rats (OVXE2), and allocated to receive liquid diet containing 5% w/v ethanol or isocaloric diet for 13 weeks. In OVXE2 but not OVX rats, ethanol feeding significantly increased renal angiotensin II level, and protein expressions of angiotensin converting enzyme, AT1 receptors, eNOS, and nNOS. Enalapril (10 mg/kg i.p) caused similar reductions in BP in OVX and OVXE2 rats. The hypotensive effect of enalapril was significantly increased in OVXE2/ethanol, but not in OVX/ethanol rats. The augmented enalapril‐evoked hypotension in OVXE2/ethanol rats was still evident in the presence of 1400W (iNOS inhibitor), but it was virtually abolished by prior inhibition of eNOS (L‐NIO), or nNOS (NPLA), or blockade of bradykinin B2 receptors (B2R, bradyzide). Together, RAS upregulation, along with facilitated B2R/eNOS/nNOS signaling, underlie the estrogen‐dependent enhancement of enalapril‐evoked hypotension in ethanol‐fed rats.
cAMP‐signaling via EPAC1 mediates vascular endothelial cell adaptation to fluid‐shear stress
Authors: Rampersad Sarah , Hubert Fabien , Umana Maria , Freitag Silja , Butler Nathalie , Brzezinska Paulina , Wudwud Alie , Maurice Donald
Published: June 2021

Herein, we report a novel role for cAMP‐signaling in human aortic endothelial cell (HAEC) adaptation to fluid‐mediated shear stress (FSS). Our results showed that increases in cAMP enhanced elongation and alignment of HAECs cultured under LSS for 48hrs and that this effect was largely mediated by the cAMP‐effector molecule, exchange protein activated by cAMP (EPAC)‐1, but not Protein Kinase A (PKA). Additionally, major HAEC cAMP‐hydrolyzing enzyme, phosphodiesterase (PDE)4D, was also shown to play a crucial role in integrating FSS signals in these cells.

Our results demonstrated that flow‐mediated increases in atheroprotective genes, Kruppel‐like factor, endothelial nitric oxide synthase and thrombomodulin mRNA expression were reduced in EPAC‐1 and PDE4D knockdown HAECs exposed to both LSS and HSS. Furthermore, EPAC‐1 knockdown increased leukocyte extravasation across HAEC monolayers in vitro and activation of EPAC significantly reduced LPS‐induced leukocyte rolling flux in mice.

VECs have numerous mechanosensors which facilitate the integration of FSS signals from the flowing blood. One such sensor consists of platelet‐endothelial cell adhesion molecule (PECAM)‐1, vascular endothelial‐cadherin (VECAD) and vascular endothelial growth factor receptor (VEFGR)‐2. Our current data suggests that EPAC1 and PDE4D can additionally mediate flow‐sensing through this mechanosensory complex. All together, this data provides important mechanistic insight providing therapeutic targets in endothelial dysfunction and atherosclerosis.
Tideglusib, an allosteric inhibitor of Glycogen Synthase Kinase (GSK)‐3β increases Angiogenic activities of Endothelial Cells
Authors: Baruah Jugajyoti , Wary Kishore
Published: June 2021

Rationale

The regulation of blood vessel growth is of paramount importance in ischemic cardiovascular and peripheral limb diseases. However, there is no effective pharmacotherapy to induce rapid revascularization of ischemic tissues.

Goal

To address if newly discovered allosteric inhibitor of GSK‐3β enzyme, Tideglusib (TDG) regulates angiogenic phenotypes of endothelial cells.

Methods and Results

The treatment of cultured ECs with Wnt3a or TDG stabilized β‐catenin polypeptide species in a time dependent manner. Quantitative RT‐PCR showed increased Cyclin‐D1, Nanog, c‐myc and Vascular Endothelial Growth Factor Receptor‐2(VEGFR‐2) transcripts. Accordingly, TDG increased expression of not only transcription factor Nanog, but also Cyclin‐D1 and VEGFR‐2 proteins. Microscopic analyses of ECs stained with anti‐VE‐cadherin antibody provided evidence of the disappearance of VE‐cadherin from the adherens junctions, while increased accumulation of β‐catenin in the nucleus. Pretreatment of ECs with TDG followed by Propidium iodide (PI) and Annexin‐V labeling showed increased survival and decreased cell death (p<0.05). Importantly, TDG treatment and the addition of 5‐bromo‐2′‐deoxyuridine (BrdU) followed by microscopy showed significant increase in the incorporation of BrdU in ECs. In addition, in a scratch assay and Boyden‐chamber cell migration experiments indicated the ability of TDG to induce migration of ECs. Furthermore, in an aortic ring assay TDG increased angiogenesis significantly.

Thus, for the first time, we show that TDG up‐regulates expression of Nanog, Cyclin‐D1 and VEGFR‐2 to augment neovessel formation, a critical biological process required for re‐establishing blood flow to the ischemic tissues.
The Contribution of Adenosine Receptor Subtypes to Vascular Tone in Mouse Pudendal Artery
Authors: Labazi Hicham , Tilley Stephen , Ledent Catherine , Mustafa S Jamal
Published: June 2021

Activation of adenosine receptors (ARs) has been implicated in a number of biological functions including cardiovascular, renal and erectile function. Recent studies suggested that only A2B AR may play an important role in penile tissue to regulate erectile function. However, no studies have been conducted on the vasculature supplying and controlling blood flow to the penis. Our aim was to determine the contribution of ARs in the regulation of vascular tone in pudendal artery (PA), the major artery supplying blood flow to the penis. Concentration response curves to non‐selective AR agonist NECA were performed in PA isolated from WT, A2A and A2B AR KOs. Our data suggested that neither A1 nor A3 AR play an important role in PA vascular tone, while genetic deletion or pharmacological inhibition of both A2A and A2B ARs significantly decreased the NECA‐mediated relaxation. Treatment of A2AKO or A2BKO PA rings with either A2B or A2AAR antagonists respectively abolished NECA‐mediated relaxation. Pre‐incubation of PA rings with L‐NAME resulted in a significant decrease in NECA‐mediated relaxation. Together, our data suggest that AR‐mediated vasorelaxation is mediated by both A2A and A2BAR in PA and is partially dependent upon nitric oxide. (Supported by HL 094447, HL027339 and HL‐071802).
AP2M1 induced internalization of TRPC6 regulates calcium influx
Authors: Piplani Honit , Sukriti Sukriti , Sharma Tiffany , Meyer Otto , Mehta Dolly
Published: June 2021

Transient receptor canonical channel (TRPC)‐6, activated by second messenger DAG, induces calcium entry in endothelial cells independent of ER store depletion. We recently showed that deletion of TRPC6 in mice prevented lung endothelial permeability and inflammation in response to thrombin and LPS indicating targeting TRPC6 will be valuable in preventing uncontrolled increase in endothelial permeability. Here we determine the pathway that inhibits TRPC6 channel activity under physiological setting. Amino acid sequence analyses of TRPC6 reveals the presence of 4 ankyrin‐like repeat domains which are known to be important for protein‐protein interactions and activity. Thus, we performed mutational analysis on these repeats and assess channel activity. We found that deletion of Ist ankyrin repeat in TRPC6 blocked calcium influx in HEK cells. Further mutational analysis identified that 96‐111 residues within Ist ankyrin repeat was required for TRPC6 cell surface retention and calcium influx. Sequence analysis using ELM database showed that this domain has consensus site for AP2M1 (Adaptor‐related protein complex 2, mu 1 subunit) binding. AP2M1 is known to internalize transmembrane proteins via clathrin dependent endocytosis. Overexpression of AP2M1 with wt‐TRPC6 in HEK cells decreased calcium influx as well as expression level of TRPC6. Thus, designing a peptide against AP2M1 binding motif in TRPC6 will help in identifying targets for inhibiting TRPC6 activity.
ADAM 17 Regulates S1PR1 Surface Expression by its Ectodomain Shedding thereby Disrupting Endothelial Barrier Function
Authors: Uddin Mohammad , Sukriti S , Desai Bhushan , Malik Asrar , Ludwig Andreas , Mehta Dolly
Published: June 2021

Acute lung injury (ALI) is characterized by excessive transendothelial leakage of protein rich fluid and infiltration of leukocytes into the alveolar space. Sphingosine phosphate receptor 1 (S1PR1), a G protein coupled receptor abundantly present in endothelial cells (ECs), upon binding to sphingosine 1 phosphate (S1P) maintains EC barrier function and inhibit leukocyte infiltration into the interstitium thereby preventing ALI. However, it is unclear whether alteration of S1PR1 expression sets the stage for induction of ALI. ADAM belongs to family of disintegrin and metalloproteinase family of enzymes which induces the shedding of the growth factor receptors and cell surface proteins. We show here that activation of ADAM in human pulmonary arterial endothelial cells (HPAECs) using phorbol 12‐myristate 13‐acetate (PMA) degraded S1PR1 and these cells failed to enhance endothelial barrier function with S1P. Among different ADAMs, ADAM 17 and ADAM 10 are known to be induced upon ALI but whether they alter S1PR1 function is unknown. Thus, we co‐transfected CHO cells with either ADAM 17 or ADAM 10 along with S1PR1. We show that ADAM 17 can cleave S1PR1 leading to S1PR1 degradation identifying for the first time S1PR1 as a substrate for ADAM17. Our data suggest that LPS destabilize endothelial barrier in part by inducing S1PR1 degradation via ADAM 17 leading to ALI.
Acid Inhibitors Exacerbate H.pylori‐Induced Gastritis Both In Vitro And In Vivo
Authors: Yanaka Akinori
Published: June 2021

Background

Gastric cancer (GC) is strongly associated with H.pylori infection. But, GC tends to develop in hypochlorhydric, but not in hyperchlorhydric patients. Since potent acid inhibitors, such as proton pump inhibitors (PPI) are widely used for the treatment of GERD, it seems reasonable to assume that potent acid inhibitors may excerbate H.pylori‐induced gastritis, promote gastric atrophy, and increase risk of GC. Based on these hypothesis, we examined (1) if the increased gastric luminal pH enhances toxicity of H.pylori‐derived ammonia in vitro, (2) if PPIs exacerbates gastritis in H.pylori‐infected mice in vivo.

Methods

A:In vitro study: 1. Intact sheets of in vitro bullfrog gastric mucosa were incubated in Ussing chambers in vitro. Transmucosal electrical resistance (TER) was monitored to evaluate mucosal integrity. 2. GSM06 cells, derived from murine gastric mucosa, were cultured with or without 5 mM NH4Cl. Cell viablity and gastric mucosal apoptosis were evaluated by MTT, and by capase‐3 assay, respectively. B: In vivo study: Six‐wk‐old female mice were cultured for 8 wks after inoculated with H.pyloi, SS‐1. Degree of gastritis was evaluated using updated Sydney sysytem. Colonization of H.pylori was also examined.

Results

A‐1. NH4Cl decreased TER at high luminal pH (>7.0), but not at low luminal pH (5.0). A‐2. NH4Cl decreased cell viality and enhanced apoptosis more at higher ambient pH (7.8), than at low pH (7.2). B‐1. PPI Tx exacerbated inflammation in corpus, but not in antral mucosa in H.pylori‐infected mice. B‐2. PPI Tx increased colonization of H.pylori only in corpus.

Conclusions

High ambient pH induced by potent acid inhibitors enhances toxicity of H.pylori‐dervied ammonia, thereby exacerbates H.pylori‐induced gastritis. Potent acid inhibition may increase risk of GC in patients with H.pylori infection.
The Role of Toll‐Like Receptor 4 in Enteric Glia
Authors: Bhave Sukhada , Brun Paola , Dewey William , Akbarali Hamid
Published: June 2021

Toll‐like receptors (TLRs) are a class of pattern recognition receptors that play an important role in mediating inflammatory responses to pathogens. TLR4 recognizes lipopolysaccharide (LPS), a membrane component of gram negative bacteria. In the GI tract, neuronal integrity may be dependent on basal activation of TLR4 receptors suggesting a protective role for TLR4. A compromised gut epithelial barrier leads to enhanced entry of bacterial proteins into the lamina propria, leading to neuronal toxicity. Thus pro‐survival vs pro‐toxic effects of LPS may be concentration‐dependent. Enteric glia are important for the maintenance of neuronal integrity. We determined the expression of pro‐inflammatory cytokines in rat enteric glia cells in response to low and high concentrations of LPS. LPS dose‐dependently increased mRNA expression of IL‐6 and TNF‐α with 0.1 μg/ml inducing a 25‐fold and 10‐fold IL‐6 and TNF‐α mRNA expression respectively as compared to untreated glia. A 100‐fold higher concentration of LPS (1‐100 μg/ml) also induced ATP release indicative of cell damage. Whole cell voltage clamp studies showed that this high concentration of LPS increased ATP‐induced inward currents with a corresponding increase in P2X1,2,3,4,and 7 receptor mRNA expression up to 50‐fold as compared to untreated glia. Thus low concentrations of LPS can induce cytokine release whereas high concentrations of LPS induce ATP release which increases P2X receptor activity and further enhances the release of pro‐inflammatory cytokines. Understanding the role of P2X in LPS induced enteric glia activation will point to new targets to control gut bacterial inflammation while still maintaining the beneficial effects of basal TLR4 signaling. Supported by NIH DK046367, DA024009.
Inhibition of miR122a by Lactobacillus rhamnosus GG Culture Supernatant Increases Intestinal Occludin Expression and Protects Mice from Alcoholic Liver Disease
Authors: Liu Liming , Zhao Cuiqing , McClain Craig , Feng Wenke
Published: June 2021

Alcoholic liver disease (ALD) has a high morbidity and mortality. Chronic alcohol consumption causes disruption of intestinal microflora homeostasis, dysfunction of the intestinal tight junction barrier, increased endotoxemia and eventually liver steatosis/steatohepatitis and injury. Probiotics supernatant supplementation has been shown to be a potential strategy for the treatment and prevention of ALD via restoring epithelial barrier function. Little is known about how probiotic supernatant works to increase intestinal tight junction proteins, thereby contributing to a protective effect in ALD. Here we show that chronic ethanol exposure increases intestinal miR122a expression, which decreases occludin expression leading to increased intestinal permeability, and Lactobacillus rhamnosus GG culture supernatant (LGGs) supplementation decreases ethanol‐elevated miR122a level and attenuates ethanol‐induced liver injury in mice. Similar to the effect of ethanol exposure, overexpression of miR122a in Caco‐2 monolayers markedly decreases occludin protein level. LGGs supplementation functions in intestinal integrity by inhibition of miR122a, leading to occludin restoration in mice exposed to chronic ethanol treatment.
Dysfunctions of the Abdominal Aorta and Renal Arteries in a Mouse Model of Neurofibromatosis Type 1
Authors: Jett Kimberly , Chohan Harleen , Cui Jason , Friedman Jan , van Breemen Cornelis , Esfandiarei Mitra
Published: June 2021

Neurofibromatosis 1 (NF1) is an autosomal dominant disorder presented with multiple café‐au‐lait spots, Lisch nodules,multiple nerve sheath tumors, and cardiovascular complications such as ischemia or aneurysm. Multiple vessels are affected in NF1 patients, but the abdominal aorta (AA) and renal arteries (RA) are most commonly reported, even though, the pathogenesis is not well understood. In this study, we examined functions of the AA and RA in a mouse model of NF1. We report a decrease in contraction and an increase in relaxation of smooth muscle cells (SMCs) in both the AA and RA at the age of 6 months. The nitric oxide synthase inhibitor (L‐NAME) completely blocked SMCs relaxation, indicating that the alterations resulted from enhanced nitric oxide (NO) production. The constitutional production of NO may reduce the availability of cofactors required for endothelial NOS to function. To determine if the increased production of NO had caused further dysfunction over time, we examined the AA in 9‐12 month old mice. Interestingly, we observed increased contraction and reduced relaxation in 9‐12 month old Nf1+/‐mice compared to controls, which may be indicative of a further decrease in NO bio‐availability. Taken together, our data demonstrates that underlying changes in SMCs and the endothelium contribute to vascular complications in NF1, providing new insights on potential future therapeutic approaches to address vascular complications in these patients. This study was funded by the British Columbia Neurofibromatosis Foundation and the Children's Tumor Foundation.
A novel peptide that directs chemotherapy against breast cancer stem cell
Authors: Lee Andy , Lee Te‐Wei , Yu John , Wu Han‐Chung , Yu Alice
Published: June 2021

L‐peptide was previously selected for its ability to bind nasopharyngeal cancer through unknown targets. Herein, we identified GRP78 as the target protein of L‐peptide and demonstrated their direct interaction using purified cell surface form of GRP78. Interestingly, L‐peptide displayed a broad spectrum of binding to a wide variety of cancers including breast cancers. Importantly, binding of L‐peptide to breast cancer clinical specimens and breast cancer stem cells (BCSC) (CD44+/CD24‐) prompted us to investigate its potential as a targeting agent for delivering chemotherapeutic agents in vivo. Treatment of mice bearing breast cancer patient derived xenografts (PDX) with L‐peptide‐conjugated lipodox (LD‐L) resulted in significantly greater suppression of tumor growth than lipodox (LD). Analyses of residual tumors showed that LD‐L but not LD reduced the BCSC subpopulation. Treated tumor cells were re‐implanted into mice to create a second‐generation model of PDX (2NDPDX) that were subsequently subjected to the same treatment regimen. Notably, 2NDPDX treated with LD‐L showed not only further suppression in tumor growth rate, tumor size, mammosphere formation but also significantly delayed the initiation of tumor when compared to that of 2NDPDX treated with LD or PBS. Most importantly, BCSC subpopulation was further reduced in the LD‐L‐treated 2NDPDX. Moreover, L‐peptide‐conjugated liposomes encapsulating isotope 188Re was able to detect tumor on xenograft models using microSPECT. Taken together, our results implicated L‐peptide as a potential cancer‐targeting agent for chemotherapy, tumor imaging, and radiotherapy.
Leishmanial Lipid Induces Apoptosis in Acute Myeloid Leukemia Cells via Regulating MAPK Signaling
Authors: Chatterjee Nabanita , Das Saha Krishna
Published: June 2021

Very promising approaches towards the microbes, its cellular component(s) or metabolites are exposed as new therapeutic entities. Among several microbial component(s), leishmanial lipids (pLLD) isolated from L. donovani has emerged for its biological potential. The lipid isolated from L. major was reported to act by inhibiting NO production in stimulated macrophages. Earlier, we have reported that pLLD possessed anti‐inflammatory activity in vitro and in vivo. Now, the determinants of differentiation and apoptosis in myelo‐monocytic leukemia (U937) cells exposed to bio‐active pLLD have been examined. This pLLD mediated apoptosis was occurred within the G0/G1 and S phase populations with altered in mitochondrial membrane potential and caspases activation, alteration of pro and anti‐apoptotic proteins and PARP‐I and other tumor suppressor protein to regulate the cellular growth. Western blot analysis showed that pLLD increased phosphorylation of ERK1/2, p38‐MAPK and JNK1/2 in concentration and time dependent manner with the cross talking of transcriptional factor STAT3 with phosphorylated subunit by flow cytometrical and immunochemical analysis. Interestingly, it reduced the elevated level of cytokines and inflammatory mediators' expression in leukemic cells. Taken together, these findings suggest that pLLD may be used as novel therapeutic agent in regulating leukemia cells growth via MAPK pathway.
Endothelial FoxO Proteins Regulate Obesity Associated Skeletal Muscle Capillary Rarefaction
Authors: Nwadozi E , Roudier E , Rullman E , Gustafsson T , Haas T
Published: June 2021

Obesity is associated with skeletal muscle capillary rarefaction, which affects muscle function and whole body metabolism. Endothelial forkhead box O (FoxO) transcription factors restrain skeletal muscle angiogenesis in response to chronic endurance exercise and during ischemic recovery. We hypothesized that FoxO proteins contribute to obesity‐related capillary rarefaction. Three PolyI:C injections were administered to induce an endothelial directed FoxO1/3a/4deletion in Mx1‐Cre+;FoxO1/3a/4L/L mice (FoxOΔ) but not in control Mx1‐Cre‐;FoxO1/3a/4L/L littermates (FoxOL/L). After 16 weeks on a high‐fat (HF) or normal chow (NC) diet, protein levels of FoxO1 (HF: 1.2±0.1 vs. NC: 0.8±0.1) but not FoxO3a (HF: 0.8±0.1 vs. NC: 0.6±0.1) were elevated in the gastrocnemius muscle. HF feeding correlated with a decreased capillary‐to‐fiber ratio in FoxOL/L (NC: 2.0±0.04 vs. HF: 1.8±0.03) but not FoxOΔ mice (NC: 2.1±0.07 vs. HF: 2.1±0.05). HF fed FoxOΔ mice gained significantly less weight compared to FoxOL/L mice (11.7±1.0 vs. 17.2±1.2 g). Insulin sensitivity was improved in HF fed FoxOΔ compared to FoxOL/L mice (plasma glucose AUC during insulin tolerance test: 23.6±2.1 vs. 37.5±5.8). Analysis of existing GEO datasets from NC and HF mouse muscle established ontology enrichment of angiogenesis‐related pathways among genes that correlated inversely with FoxO1 expression. Thesefactors, putatively repressed by FoxO1, are postulated to be differentially expressed in FoxOΔ and FoxOL/L mice. These results suggest that endothelial FoxO proteins contribute to obesity related skeletal muscle capillary rarefaction, and the associated metabolic phenotypes of weight gain and impaired insulin sensitivity. Funded by CIHR.
p18 Regulates Tubulogenesis in the Pulmonary Endothelium Through p38/mTOR‐dependent Pathway
Authors: Chichger Havovi , Braza Julie , Duong Huetran , Stark Myranda , Harrington Elizabeth
Published: June 2021

Vasculogenesis and angiogenesis, the formation of new blood vessels, are processes requiring multiple endothelial cell (EC) functions, including proliferation, migration and adhesion. Blood vessel lumen formation is necessary for the creation of a functional vessel to transport blood, fluids, and nutrients and occurs via tubulogenesis. This process of lumen formation is not completely understood, however there is evidence for the role of the endosome. The endosomal adaptor protein p18 anchors the late endosome/lysosome to facilitate mTOR/MAPK activation. We have demonstrated that p18 binds to the early endosome to enhance VE‐cadherin recycling to EC junctions and improve EC barrier function via an mTOR/MAPK independent pathway. As EC permeability is a key component of tubulogenesis, we hypothesize that p18 localized to the endosome regulates tubulogenesis in the pulmonary EC, potentially through regulation of MAPK and mTOR activity. Lung EC were transfected with cDNA encoding p18wt display significantly increased EC migration, adhesion to gelatin and in vitro and in vivo tube formation, but had no effect of proliferation. Inhibition of mTOR (rapamycin) or p38MAPK (SB203580) significantly attenuated the pro‐tubulogenic role of p18wt. Conversely, overexpression of the non‐endosomal binding p18 (p18N39) and p18 siRNA knockdown attenuated VEGF‐induced adhesion and proliferation. Thus our data demonstrates a key role for endocytic protein p18 in promoting tubulogenesis within the pulmonary vasculature through activation of mTOR and p38. Thus we implicate p18 as a novel therapeutic target to regulate vessel formation within the pulmonary vasculature.
Flow Regulates Thrombin Generation Kinetics
Authors: Yin Wei , Bond Kyle , Musillo Michael , Rubenstein David
Published: June 2021

Shear stress plays important roles in platelet coagulation. However, in many studies, coagulation rate constants were determined under static conditions. In the present study, we aimed to determine how flow affects thrombin generation rates, when coagulation factors were immediately available. Washed platelets were exposed to various dynamic shear stress (physiological and pathological) in a cone and plate shearing device, with Factor Xa, prothrombin, and calcium. Thrombin generation was quantified using chromozym‐TH absorbance at 405 nm. Platelet activation was assessed by cell surface P‐selectin expression using flow cytometry. The results demonstrate that elevated dynamic shear stress induced a significant increase in P‐selectin expression, indicating platelet activation. However, thrombin generation rate was significantly reduced under elevated shear stress, compared to that under low or physiological shear stress. Such results were different from that observed with a prothrombinase assay following platelet exposure to dynamic shear stress. The immediate availability of coagulation factors likely contributed to the observed differences. These data suggest that dynamic shear stress affects thrombin generation kinetics, not only by affecting platelet activation state, but also by affecting prothrombinase complex formation on platelet surface. Thrombin generation rates could be reduced under accelerated flow and elevated shear stress, as reaction time for prothrombinase complex to form is significantly reduced.
Endothelial Glycocalyx and Apoptosis in Atherosclerosis
Authors: Cancel Limary , Ebong Eno , Tarbell John
Published: June 2021

Elevated permeability of vascular endothelium to low density lipoprotein (LDL) is characteristic of focal sites within the vasculature that are prone to the development of atherosclerosis. Previous experiments have suggested that both increased endothelial cell (EC) apoptosis and endothelial surface glycocalyx (GCX) shedding could play a role in the elevated LDL permeability of athero‐prone regions. In this work, we sought to confirm that GCX shedding and increased EC apoptosis do occur in lipid plaque filled vessel areas. Six 4‐ to 6‐week old male C57Bl/6 apolipoprotein E knockout (ApoE) mice were fed a Western diet for 10 weeks and developed raised plaques in their brachiocephalic arteries (BCA). Our results show that EC apoptosis is significantly increased throughout the vasculature of ApoE mice compared with wild type mice fed a normal diet. More importantly, we demonstrate that the number of apoptotic ECs per unit length is 3 times higher in the plaque region of the BCA of ApoE mice than in the non‐plaque region of the same vessel. We also show that GCX coverage and thickness are significantly reduced over the plaque region compared to the non‐plaque region, where values are not different from those found in the wild type mice. Our results suggest that both increased EC apoptosis and GCX are involved in the development of atherosclerotic plaques. Research supported by NIH HL094889.
INSULIN‐LIKE GROWTH FACTOR I (IGF‐1) REDUCES LIPID OXIDATION AND FOAM CELL FORMATION VIA DOWNREGULATION OF 12/15‐LIPOXYGENASE
Authors: Sukhanov Sergiy , Snarski Patricia , Kim Catherine , Delafontaine Patrice
Published: June 2021

We have previously shown that IGF‐1 decreased atherosclerosis, plaque lipids and macrophages (MF) suggesting suppression of MF‐derived foam cells. Since 12/15‐lipoxygenase (LOX) promotes LDL oxidation/formation of foam cells, we hypothesized that IGF‐1 downregulates LOX and via this mechanism reduces foam cells. IGF‐1 infusion (1.5 mg/kg/d) in Apoe‐/‐ mice fed a Western diet decreased plaque LOX protein (IHC, 67±6% decrease) and aortic LOX mRNA (36±4% decrease). IGF‐1 reduced serum oxidized LDL and MDA levels (a marker of lipid oxidation) (19.7% and 22% decrease, respectively), indicating that IGF‐1 suppressed lipid oxidation in vivo. IGF‐1 (50 ng/ml, 24h) reduced LOX protein and mRNA levels in cultured human THP‐1 MF (49±5% and 48±6% decrease, respectively) and IGF‐1 decreased expression of STAT‐6 transcription factor (38±4% decrease). Anti‐STAT6 siRNA reduced both STAT6 and LOX levels indicating STAT6 involvement in LOX transcriptional regulation. IGF‐1‐induced LOX decrease was blocked by PI3 kinase inhibition (LY2940032, 50 uM). IGF‐1 suppressed the ability of THP‐1 MF to oxidize lipids (TBARS assay, 65±4% decrease) and to form foam cells (Oil Red staining, 64±4% decrease). Peritoneal MF from LOX‐/‐ mice had reduced ability to oxidize lipids and form foam cells vs. WT MF. IGF‐1 decreased LDL oxidation and foam cells in WT MF but these effects were blocked in LOX‐/‐ MF. In summary, we shown that IGF‐1 decreased LOX via a PI3 kinase‐ and STAT6‐dependent mechanism, resulting in reduced MF‐mediated LDL oxidation and foam cell formation. IGF‐1‐induced and LOX‐dependent suppression of foam cells potentially plays a major role in the ability of IGF‐1 to exert anti‐atherosclerotic effects.
Maximizing the Max Test: A Method for Determining Cardiorespiratory Fitness and Dysfunction in Mice.
Authors: Petrosino Jennifer , Mauyra Santosh , Kalyanasundaram Anuradha , Wilson Jacob , Periasamy Muthu , Ziouzenkova Ouliana
Published: June 2021

Early diagnostic of cardiovascular morbidity plays a critical role preventing mortality. The Bruce protocol is an exercise diagnostic test that can determine cardiorespiratory fitness. This is a standardized protocol to evaluate cardiac function in humans, yet no such standardize test has been developed for widespread use with animal models.

PURPOSE

The purpose of the study is to develop a similar method of testing V02max in mice. METHODS: Male WT mice (C57BL06, n=7) and mice with impaired cardiovascular function (Casq2‐null and obese WT, n=15) were used to optimize a progressive speed run test vs. our graded maximal run test and identify critical parameters indicating cardiovascular dysfunction.

RESULTS

Our graded test compared to the progressive lead to increased VO2max in reduced time in obesity model. With WT and Casq2‐null similar V02max was achieved in less time. Our graded test allowed for determination of a metabolic crossover parameter occurring sooner in dysfunctional animals compared to controls, as well as differential points of ventilatory threshold (VAT), time to VO2max, and %VO2max at VAT (P<.05). We determined that the crossover parameter is the most accurate predictor of cardiac dysfunction.

CONCLUSIONS

The graded exercise protocol provided valuable metabolic and cardiovascular information for fast and noninvasive evaluation of cardiovascular dysfunction in animal models.
Effects of arginine vasopressin deficiency on innate immunity in the rat
Authors: Quintanar‐Stephano Andrés , Viñuela‐Berni Verónica , Organista‐Esparza Alejandro , Tinajero‐Ruelas Manuel , Huerta‐Carreón Erika , Kovacs Kalman , Berczi Istvan
Published: June 2021

Immune and vascular endothelial cells possess arginine vasopressin (AVP) receptors. AVP is an important stimulating/regulatory hormone in acquired immunity, whereas its role on innate immunity (INIM) is not well known. Neurointermediate pituitary lobectomy (NIL) in the rat causes permanent low AVP serum levels. Here, we investigate the AVP and INIM relationship in NIL rats (3 weeks after surgeries) subject to INIM tests: Phagocytic index (PI) (peritoneal macrophages (Mθ) erytrophagocytosis), and Skin Evans blue extravasation‐histamine doses‐response test. In the Study 1, NIL group was compared against Intact control (IC), sham operated (SHAM) and anterior pituitary lobectomysed (AL) groups. In the study 2, NIL group was compared against IC, NIL+desmopressin (a synthetic analog of AVP) and IC+conivaptan (antagonist of V1a‐V2 AVP receptors) groups. Results: Study 1 showed that as compared with the IC and AL groups, PI was significant decreased in NIL animals. In the Evans blue extravasation‐histamine test study, a significant and similar increases of skin edema histamine doses‐dependent occurred in NIL and IC+conivaptan groups, whereas no significant differences in IC and NIL+desmopressin groups were appreciated.

Conclusion

AVP plays an important role in phagocytic activity of the peritoneal Mθ and for the stabilization of the vascular endothelial cells permeability during histamine inflammation.

Supported by UAA‐PIFF 14‐1. Universidad Autónoma de Aguascalientes, México and the Jarislowsky and Lloyd Carr‐Harris Foundations.
The Cardioprotective Effects of a NOX1 Inhibitor, ML171, on Myocardial Ischemia/Reperfusion (I/R) injury
Authors: Remarcke Ryan , Patel Shan , Stutzman Devon , Ondrasik Regina , Devine Issachar , Patel Harsh , Chau William , Patel Samir , Peters Colin , Smith Edward , Pang Clifford , Barsotti Robert , Young Lindon , Chen Qian
Published: June 2021

Oxidative stress is a major cause of I/R injury. NADPH oxidase is an important source of oxidative stress during I/R, and it consists of NOX1‐5 and DOUX1‐2 isoforms. The role of NOX1 in myocardial I/R injury is unclear. In this study, a specific NOX1 inhibitor, ML171 (MW=241.31 g/mol, IC50 for NOX1=0.25 μM), was tested in isolated perfused rat hearts following I (30 min)/R (45 min). We found that left ventricular developed pressure (LVDP) and the peak of the first derivative of left ventricular pressure (dP/dtmax) only recovered to 42 ± 4% and 30 ± 3% of baseline values, respectively, at 45 min post‐reperfusion in control I/R hearts (n=8). By contrast, ML171 (1 μM, n=5) given at reperfusion for 5 min significantly restored LVDP and dP/dtmax to 90 ± 6% and 67 ± 7% of baseline values, respectively (both p<0.01). ML171 also significantly reduced infarct size to 18 ± 3% compared to 41 ± 2% in control I/R hearts (p<0.01). Furthermore, a selective mitochondrial ATP‐dependent K+ channel (mitoKatp) inhibitor, 5‐ hydroxydecanoate (100 μM, n=4); and a heme oxygenase‐1 inhibitor, SnPP (20 μM, n=3), significantly abolished the cardioprotective effects of ML171 (all p<0.05). We also confirmed that ML171 (0.1‐1 μM, n=4) did not inhibit phorbol 12‐myristate 13‐acetate (30 nM) induced superoxide release from isolated rat neutrophils. These results suggest that NOX1 inhibition improves post‐reperfused contractile function and reduces infarct size, possibly by opening mitoKatp channels and activating heme oxygenase‐1.
Hydrogen peroxide‐induced redox homeostasis regulation in endothelial cells
Authors: Patel Hemang , Chen Juan , Kavdia Mahendra
Published: June 2021

Human vasculature is shown to regulate hydrogen peroxide (H2O2) at various levels in normal as well as pathophysiological conditions. Recent research studies have put forward the involvement of H2O2 in regulating cytotoxic and vasoactive response to vasculature. However, being part of redox system, the effect of H2O2 on regulation of vascular and in particular endothelial redox system is not clearly understood. Thus, we investigated H2O2 –induced regulation in redox system during transient and consistent exposure to endothelial cells. Using systems biology approach, we studied the superoxide (O₂‾) production, mitochondrial membrane polarization and gene expression of several redox system enzymes in HUVEC following both transient and consistent H2O2 exposure for 72 hours. Our results, showed a significant down‐regulation in ASK1, CAT, TXNRD1 under continuous H2O2 exposure. But in transient H2O2 exposure followed by remodeling period showed significant up‐regulation in GPX1 and TXNRD1 whereas genes expression of ASK1, SOD1 and PRDX1 returned to homeostasis. In both treatment conditions, exposure to H₂O₂ significantly increased O₂‾ production, which remained significantly higher up to 48 hours and decreased back to control level. Similar trend was also observed in mitochondrial membrane polarization which transitioned from hyperpolarized to normal state over the period of 72 hours in both H₂O₂ treatment conditions. Results from our study indicate that HUVEC actively regulated functional and gene expression changes to reach similar extent of redox homeostasis under both H₂O₂ treatments. These results further the understanding on how endothelial cells endure varying levels of H₂O₂ in pathological conditions.
The Effects of Bilirubin on Angiogenesis in Mice with Hindlimb Ischemia
Authors: Ikeda Yasumasa , Kanai Yusuke , Horinouchi Yuya , Izawa‐Ishizawa Yuki , Kihira Yoshitaka , Miyamoto Licht , Ishizawa Keisuke , Tsuchiya Koichiro , Tamaki Toshiaki
Published: June 2021

Background

Low serum bilirubin levels are associated with peripheral artery disease. Bilirubin is known to exert anti‐oxidant effect and the enhancement of flow‐mediated vasodilation, however, bilirubin action on angiogenesis remains unclear. In this study, we examine the mechanism of bilirubin on angiogenic effect.

Methods and Results

We employed C57BL/6J mice with unilateral hindlimb ischemia surgery and divided the mice into two groups (vehicle‐treated group and bilirubin treated group). The analysis of laser speckle blood flow demonstrated that the recovery of blood flow in response to ischemic hindlimb was promoted in mice with bilirubin treatment. The capillary density was significantly higher in ischemic lower limb of bilirubin‐treated mice. The phosphorylated levels of endothelial nitric oxide synthesis (eNOS) and Akt were augmented in ischemic tissues of mice with bilirubin treatment compared vehicle treatment. In in vitro experiments by using human aortic endothelial cells, bilirubin increased eNOS and Akt phosphorylation. Bilirubin also augmented cell proliferation, cell migration and tube formation. These bilirubin action on endothelial cell activation were abolished by LY294002, a phosphatidylinositol 3‐kinase inhibitor.

Conclusion

Bilirubin promotes angiogenesis through endothelial cells activation via Akt‐eNOS‐dependent axis.
Characterization of Lung‐specific Angiogenesis
Authors: Mammoto Akiko , Jiang Amanda , Jiang Elisabeth , Mammoto Tadanori
Published: June 2021

Although recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate simple tissues in the orthopedic and periodontal fields, attempts to regenerate the structures and functions of more complex organs such as lungs have not been very successful. This may be partly because the biological processes of organ regeneration have not been well explored. Angiogenesis, the formation of new blood vessels, in specific organ plays key roles in organ regeneration. Therefore, the goal of this project is to recapitulate the lung‐specific microenvironments and replicate angiogenesis in a specific organ in situ in order to regenerate lungs in mice. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix‐rich hydrogels (e.g., Matrigel), are extensively utilized to explore the general mechanisms of angiogenesis, lung‐specific angiogenesis has not been well characterized because the methods for implantation of biomaterials on the mouse lung have not been well established. Here we have developed a unique method to implant fibrin gel on the lung surface of living mouse, allowing for the successful recapitulation of host lung‐derived angiogenesis inside the gel. We also found that tumor‐derived angiogenic factor induced angiogenesis in the lung using a similar implantation method. Thus, this approach enables researchers to explore the mechanisms by which the lung‐specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. This project is supported by American Heart Association and Boston Children's Hospital Career Development Award.
Sildenafil Improves Flow‐Mediated Dilation in Adults with Cystic Fibrosis
Authors: Harris Ryan , Seigler Nichole , Thomas Jeff , McKie Kathleen , Forseen Caralee
Published: June 2021

Our group has provided evidence that endothelial function, assessed by the flow‐mediated dilation (FMD) test, is attenuated in patients with Cystic Fibrosis (CF) compared to healthy controls. The mechanisms for the dysfunction; however, have yet to be elucidated. Phosphodiesterase type 5 (PDE5) inhibitors have been shown to improve endothelial function in different patient populations, thus this study sought to determine if treatment with sildenafil improves FMD in patients with CF. To date we have recruited 4 adult patients with CF (2 men and 2 women; 35±4 years, mean FEV1 67±10 % predicted), 3 of whom have completed the protocol. Brachial artery FMD was performed at baseline and 4 weeks following treatment with sildenafil (20 mg, 3 x daily). Baseline brachial artery diameter increased (p=0.09) following 4 weeks of sildenafil (0.287 ± 0.04 cm to 0.296 ± 0.03 cm, respectively). FMD expressed as absolute change in diameter was significantly (p=0.04) improved from baseline (0.019 ±0.005 cm) to 4 weeks (0.026±0.006 cm), whereas relative FMD(%) was improved (p=0.09) following treatment (6.1±1.2% to 8.4±0.9%, respectively). No differences in shear rate (p=0.413) or time to peak dilation (p=0.463) were observed following treatment. Although the study has just begun, provocative preliminary findings suggest that 4 weeks of sildenafil can improve endothelial function in patients with cystic fibrosis. PDE5 inhibition may represent an adjunctive therapy to improve endothelial function in patients with CF. Supported by NIH/NIDDK R21DK100783 (RAH).
H2S‐tolerant Fish Differentially Express Genes and Modify Mitochondrial Function to Maintain H2S Homeostasis
Authors: Shaw Jennifer , Henpita Chathurika , Tobler Michael
Published: June 2021

Hydrogen sulfide (H2S) is a toxic gas that inhibits the final step in the mitochondrial electron transport chain. This gas, however, is produced endogenously at low levels to promote vasodilation, angiogenesis, and reduce inflammation. Both a deficiency and an excess of H2S are associated with disease therefore, maintaining H2S homeostasis is critical for proper physiologic function. Despite the surge in biomedical interest in H2S as a therapeutic and a factor in the etiology of disease, the regulatory mechanisms remain largely unknown. Progress has been hampered by the lack of study systems that exhibit both sulfide susceptibility and sulfide tolerance within a single species that would permit comparative analyses toward uncovering mechanisms that regulate responses to H2S. Poecilia mexicana is a species of fish with populations that are naturally adapted to either non‐sulfidic or sulfidic environments. Offspring reared in the lab under non‐sulfidic conditions maintain their tolerance or non‐tolerance to H2S. We hypothesized that H2S‐tolerant fish differentially express genes and modify mitochondrial function to tolerate toxic levels of H2S relative to non‐tolerant fish. We have found that H2S‐tolerant fish differentially express SQR, VEGF, CSE, COX, and P450 (which are involved in H2S responses in other models) under control and H2S conditions relative to non‐tolerant fish. Furthermore, mitochondria isolated from H2S‐tolerant fish livers exhibit modifications in the coupling of electron transport and oxidative phosphorylation compared to non‐tolerant fish mitochondria. These data suggest that this is a valuable model to elucidate mechanisms involved in regulating H2S.
Interrelationships Between Platelet Activity and the Immunoinflammatory Response to Severe Injury
Authors: Morris Rachel , Schaffer Beverly , Lundy John , Pidcoke Heather , Cap Andrew , Schwacha Martin
Published: June 2021

Background

Critical injury causes alterations in coagulation and inflammation. Activation of these systems is critical to counteract the initial threats of hemorrhage and infection; however prolonged propagation is associated with poor outcomes. How coagulation and inflammation interact is an area of keen interest. The aim of this study is to correlate changes in circulating inflammatory mediators with coagulation in trauma (ISS>15) and burn patients.

Methods

Blood samples were drawn from trauma (n=10) and burn (n=10) patients and healthy volunteers (HV; n‐10). Coagulation parameters (sCD40L, D‐Dimers), cytokines (IL‐6, IL‐10, IL‐17a, TNFα) and inflammatory markers (HSP‐72) were assessed.

Results

The subjects were predominately male (85%) and ~44 years of age. A marked increase in IL‐6 and IL‐10 was observed in both injury groups, whereas only the trauma group showed an increase in IL‐17a and TNFα compared with HV. HSP‐72 levels were 3‐fold higher in the trauma group, but not elevated in the burn group compared with the HVs. In contrast, sCD40L, a marker of platelet activation, was elevated 10‐25 fold, and D‐Dimers were elevated 5‐8 fold in both injury groups. A strong correlation between IL‐17a and sCD40L was observed in the HVs and the burn group (R2 > 0.80), but not the trauma group (R2=0.02).

Conclusion

These results show that the post‐injury inflammatory response is paralleled by activation of coagulation. However, in trauma patients, a critical interrelationship between platelet activation and the Th‐17 response appears to be lacking, which may contribute to coagulopathic complications and warrants further study.
Role of endothelial PPARγ: Protection against vascular dysfunction induced by IL‐1β
Authors: Mukohda Masashi , Quelle Frederick , Sigmund Curt
Published: June 2021

Peroxisome proliferator‐activated receptor gamma (PPARγ) has been proposed to mediate anti‐inflammatory actions by promoting the degradation of the p65 subunit of nuclear factor kappa B (NFκB). We hypothesize that PPARγ protects against NFκB‐induced vascular dysfunction by promoting p65 degradation in vascular endothelial cells (EC). We used a model of interleukin‐1β (IL‐1β)‐induced aortic dysfunction. Aorta was treated with IL‐1β (24 hr) in vitro and then EC function was measured in an organ chamber. IL‐1β dose‐dependently decreased IκBα expression, increased phospho‐p65, and increased luciferase activity in NFκB‐Luc transgenic mice. IL‐1β dose‐dependently reduced EC‐dependent relaxation by acetylcholine (ACh). The loss of ACh responsiveness was partially improved by pretreating vessels with the PPARγ agonist, rosiglitazone, or in transgenic mice over‐expressing wildtype PPARγ specifically in EC (E‐WT). Consistent with this, low dose IL‐1β was sufficient to cause EC dysfunction in aorta from transgenic mice expressing dominant negative PPARγ specifically in EC (E‐V290M). A p65 inhibitor peptide did not alleviate EC dysfunction induced by IL‐1β, but Tempol, a superoxide dismutase mimetic partially restored ACh responsiveness in both control and E‐WT aorta treated with IL‐1β. TUDCA, a chemical chaperone which reduces endoplasmic reticulum (ER) stress significantly improved EC dysfunction induced by IL‐1β in control aorta, but not aorta from E‐WT mice. Aorta isolated from CHOP‐deficient mice exhibited less IL‐1β‐induced EC dysfunction than control mice. These data suggest that EC PPARγ protects against IL‐1β‐mediated EC dysfunction, possibly through the reduction of reactive oxygen species and blunting IL‐1β‐mediated ER stress.