bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased plasma levels of TGF‐beta1 and CD14+/CD105+ numbers in by‐pass patients correlate to increased CD105‐induced expression after plasma incubation
Authors: Tejerina Teresa , Navarro‐Dorado Jorge , Ramajo Marta , Gordillo‐Moscoso Antonio , Redondo Santiago , Carnero Manuel , Gonzalez‐Rocafort Alvaro , Alegria Victoria , Rodriguez Enrique , Reguillo Fernando
Published: June 2021

Objective

Compare the numbers of CD14+/CD105+, plasma levels of TGF‐beta1, apoptosis and induced CD105 expression in atherosclerotic patients (bypass surgery) and control ones (valvular patients).

Methods

Blood was taken from our patients and CD34+/CD144+, CD34+/KDR+ and CD14+/CD105+ cells were assessed by flow cytometry (R=0.8908). Plasma was added to peripheral blood mononuclear cells. CD105 expression was assessed by Western blot (day 4). Apoptosis was measured as DNA fragmentation (day 7).

Results

By‐pass patients had a higher number of CD14+/CD105+ (5.357 vs 2.829% lymphocytes, p=0.04; 87.13 vs 49.77 cells/mcL, p=0.0339) and a higher level of plasmatic TGF‐beta1 (72.01 vs 48.33, p=0.0033). When plasma from by‐pass and valvular was added to cell (day 7), valvular plasma triggered apoptosis (0.9998 vs 1.599, p=0.0078), an effect mimicked when plasma from by‐pass was coincubated with the TGF‐beta1 blockers SIS3 (0.9998 vs 1.624, p=0.0034) and SB‐431542 (0.9998 vs 1.802, p=0.0108). In Western blot, a trend was found towards higher CD105 expression with plasma from bypass (0.94 vs. 0.57, p=0.18).

Conclusion

Plasma from by‐pass patients deregulates the effect of TGF‐beta1, which correlates to increased plasmatic TGF‐beta1 levels and CD14+/CD105+ cells and a trend towards higher in vitro expression of CD105.

Disclosure

Funded by FISS PI041018 (Spanish Ministry of Health).
Vascular α1C and β3 subunits of L‐type calcium channels are coordinately upregulated in the mesenteric arteries of hypertensive C57BL6 mice
Authors: Kharade Sujay V , Sonkusare Swapnil , Rhee Sung W , Rusch Nancy J
Published: June 2021

Depolarization of vascular smooth muscle cells (VSMCs) opens L‐type calcium (CaL) channels to elicit Ca2+‐dependent contraction. CaL channels consist of pore‐forming α1C and one of four β subunits (β1‐4) that promote α1C expression. β3 is regarded as the major subunit in large arteries, but its functional role in resistance arteries is unclear. We hypothesized that α1C and β3 are coordinately upregulated in hypertension. We infused C57BL6 mice with Angiotensin II (Ang II, 2 ng/g/min, 2 weeks) using osmotic minipumps to establish hypertension. Systolic blood pressures (SBPs) were 170 ± 10 mm Hg in Ang II hypertensive (AHT) mice compared to 114 ± 3 mm Hg in saline infused control mice. Nifedipine (5 mg/kg, i.p.) restored SBP to normal levels in AHT but only mildly lowered SBP in control mice. Isolated mesenteric arteries (MA) from AHT mice showed a 5.1‐fold higher contractile sensitivity to the CaL channel agonist FPL64176 (n=5), and CaL channel current density was increased 1.9‐fold in the mesenteric VSMCs (n=23). Western blots revealed a dual upregulation of α1C and β3 in MA from AHT mice and a marked increase in α1C and β3 immunofluorescence at the membrane of VSMCs. Collectively, our study suggests that: i) CaL channels are primary contributors to hypertension in the AHT mouse, and ii) the α1C/β3 complexes may be the molecular pathways for anomalous Ca2+ influx and vascular tone. Supported by R01 HL064806‐07 (NJR)
Functional Implication of Lysosomal TRP‐ML1 Channel Deficiency: Response to NAADP
Authors: Zhang Fan , Li Pin‐Lan
Published: June 2021

It is well known that the mutation of TRP‐ML1 causes mucolipidosis IV, a lysosomal storage disease. Given that NAADP is activator of lysosomal TRP‐ML1 Ca2+ release channel, the present study was designed to test whether NAADP acts on TRP‐ML1 and contributes to a Ca2+‐dependent lysosome and endosome fission and thereby regulates lipid trafficking. Using lysosomal preparations from human fibroblasts, it was found that NAADP (100 nM) significantly increased the channel open probability from 0.0066±0.0018 of basal level to 0.0501±0.0073 by lipid bilayer assay, but abolished in the lysosmes from TRP‐ML1−/− cells. NAADP increased channel activity could be markedly blocked by NAADP antagonist PPADS. In intact cell fluorescence imaging, NAADP (100 nM) significantly increased the Ca2+ release from the basal level of 159.77 ± 55.46 to 287.06 ± 61.37 nM in wild fibroblasts but not in TRP‐ML1−/− cells. Functionally, confocal microscopy revealed that NAADP significantly enhanced the fusion and fission frequency of endosomes and lysosomes in wild type fibroblasts, and had no effects in TRP‐ML1−/− cells, but restored after TRP‐ML1 gene rescued. These results suggest that NAADP activates lysosomal TRP‐ML1 Ca2+ channel, which promotes a fusion‐fission process of endosomes and lysosomes, and facilitates intracellular lipid trafficking in human fibroblasts. (Supported by NIH grants HL57244, HL075316, and DK54927).
EP1 prostaoid receptor mediated upregulation of hypoxia inducible factor‐1α (HIF‐1α) under normoxic conditions by activation of Gi‐coupled phosphoinositide 3‐kinase (PI3K) signaling
Authors: Ji Ruyue , Chou Chih‐Ling , Xu Wei , Regan John W
Published: June 2021

The EP1 prostanoid receptor is one of four receptor subtypes activated by prostaglandin‐E2 (PGE2) and is known to couple to Gq to activate calcium signaling. Recently we have found that PGE2 stimulation of the human EP1 receptor can upregulate the expression of HIF‐1α under normoxic conditions. HIF‐1 is a heterodimeric transcription factor consisting of HIF‐1α and HIF‐1β. While HIF‐1β is expressed constitutively, HIF‐1α expression is typically regulated by hypoxia. Thus, during normoxia HIF‐1α is rapidly degraded, whereas, during hypoxia its degradation is inhibited: HIF‐1α then accumulates, combining with HIF‐1β to activate transcription via binding to hypoxia response elements (HRE) in HIF‐regulated target genes. We have found by Western blot analysis that the upregulation of HIF‐1α by PGE2 stimulation of the human EP1 receptor expressed in HEK cells is completely blocked by pretreatment of cells with pertussis toxin, an inhibitor of Gi/o. Functional activation of an HRE/luciferase reporter gene is also blocked by this treatment. Furthermore, pretreatment of cells with wortmannin, an inhibitor of PI3K, or with Akt‐inhibitor, decreases the PGE2 stimulated upregulation of HIF‐1α protein expression and HRE functional activity. In conclusion PGE2 stimulation of the EP1 receptor can upregulate the expression of HIF‐1α by a mechanism involving coupling of the receptor to Gi and activation of PI3K and Akt signaling.
P2Y2 receptors re‐sensitize TRPV1 via PKC activation in kidney projecting sensory neurons
Authors: Wang Hui , Wang Donna H. , Galligan James J.
Published: June 2021

The transient receptor potential vanilloid 1 (TRPV1) receptor is a ligand‐gated cation channel expressed in sensory nerves. TRPV1 regulates salt‐sensitivity, renal blood flow and blood pressure via release of calcitonin gene related peptide from sensory nerves. Sympathetic nerves are closely aligned with sensory nerves in the renal artery and sympathetic nerves release ATP that can act on P2Y receptors expressed by sensory nerves. We showed that activation of P2Y2 receptors by ATP re‐sensitizes TRPV1 in kidney projecting sensory neurons. To understand the mechanism underlying receptor interaction, the intracellular signaling pathway was studied. Fast Blue was used to retrogradely label kidney projecting sensory neurons in rat dorsal root ganglia in vivo. Ganglia were harvested and sensory neurons were maintained in short term primary culture. Whole‐cell recording studies showed that ATP or UTP, acting at P2Y2 receptors, and PMA, a protein kinase C (PKC) activator, reversed capsaicin‐induced desensitization of TRPV1. These effects were blocked by the PKC inhibitors, staurosporine and chelerythrine. These data indicate that receptor interaction between P2Y2 and TRPV1 is mediated by PKC activation in kidney projecting sensory neurons. P2Y2 receptor modulation of TRPV1 function may be a mechanism of interaction between sympathetic and sensory nerves supplying the renal vasculature.
Angiotensin II‐regulated Genes in Vascular Smooth Muscle Cells: Promoter Interactions between CREB and HIF
Authors: Lounsbury Karen M. , Rose Patricia , Sateriale Adam , Roy Daniel , Taggart Laura , Wellman Theresa
Published: June 2021

The underlying cause of genetic changes in vascular smooth muscle from hypertensive disease is uncertain. The transcription factor CREB is hyperactive in cerebral arteries from Dahl S hypertensive rats and is activated by angiotensin II (Ang II) in cultured smooth muscle cells. By using microarray analyses, we identified several CRE‐containing genes that are calcium‐dependent and/or upregulated in arteries from Dahl hypertensive rats. Our objective was to test the hypothesis that Ang II regulates these genes through complex interactions between CREB and other transcription factors. The MAP kinase phosphatase‐1 (mkp‐1) gene was chosen for analysis due to its induction by Ang II and its role in cell proliferation. Analysis of the mkp‐1 promoter revealed nucleotide overlap between its CRE sites and hypoxia response elements (HREs). Ang II promoted specific interaction between CREB and an mkp‐1 CRE/HIF promoter region, as measured by chromatin immunoprecipitation (ChIP). Knockdown of CREB using siRNA also prevented induction of mkp‐1 mRNA levels by Ang II. Hypoxic induction of HIF‐1α blunted the Ang II induction of mkp‐1 transcription, and reduced mRNA levels of CREB, suggesting that HIF‐1α may inhibit CREB‐mediated transcription. These results provide new evidence for hypoxic regulation of Ang II‐mediated gene expression. Supported by NIH HL067351 and the Totman Center for Cerebrovascular Research.
The learning deficit of the Ts65Dn mouse, a model for Down syndrome, persists with age but does not increase
Authors: Sanders Nichole Chase , Wenger Galen R
Published: June 2021

Previous research using an incremental repeated acquisition (IRA) schedule of reinforcement has shown there is a significant learning impairment in young, adult Ts65Dn mice (TS) compared to littermate controls (LC) (Wenger et al. Behav. Genet. 34: 105‐119, 2004). The purpose of this study was to determine if this deficit changes with age. A performance version of the task (IRA‐P), under which only the correct position response was illuminated and as such, is not a measure of acquisition, was used to determine if changes in the deficit were caused by changes in motoric ability. Data collected under the IRA task showed that there was a significant learning deficit that persisted up to 24‐months of age in the TS compared to LC. There was a significant decline in the rate of responding and the number of milk presentations earned by the TS after 19‐months of age. However, response accuracy, which is not dependent on mobility, did not decline. Under the IRA‐P schedule, no change was observed in the number of milk presentations of either line as they aged, but there was a declining trend in the rate of responding of the TS similar to that observed in the TS under the IRA schedule. These data indicate there is not an age related decline in the ability to learn in TS. Supported in part by NIH grant HD047656 (G.R.W.) and the NIEHS pre‐doctoral training grant 5T32ES01952.
L‐DOPA treatment modulates the expression and activity of L‐aromatic amino acid decarboxylase in the MPTP mouse model
Authors: King Jennifer , Mackey Veronica R. , Gladson Muthian , Charlton Clivel G
Published: June 2021

The replacement of dopamine (DA) with its precursor L‐dopa has remained the most effective symptomatic treatment for Parkinson's disease (PD), although the majority of patients experience a decline in efficacy as well as motor complications after prolonged usage. Although effective, L‐dopa treatment does not restore the physiological levels of DA transmission, and cycles of high and low rates of DA produced can contribute to the pathophysiology of the motor complications that occur. Our results have shown that in the MPTP‐PD mouse model that received sub‐chronic L‐dopa treatments, L‐aromatic amino acid decarboxylase (LAAD), the enzyme that converts L‐dopa to DA, was remarkably reduced in the striatum, and hyperkinesia was observed in the PD model treated with L‐dopa. Striatal tyrosine hydroxylase (TH) expression was decreased in the PD model treated with L‐dopa as well as striatal LAAD activity. DA and its metabolites levels were decreased as well in the PD model treated with L‐dopa along with a decrease in the expression of LAAD. These findings suggest that the loss of efficacy of L‐dopa could possibly be related to the down‐regulation of LAAD that was observed in the striatum, possibly by L‐dopa induced production of dopamine. This observed reduction of LAAD expression and activity is critical in L‐dopa treatment because less DA is synthesized which is needed to counteract the decrease in DA caused by the PD neurodegeneration. Therefore, a greater understanding in the modulation of LAAD could possibly prolong the efficacy of L‐DOPA therapy.
Current Drug Scheduling Reviews Reported by DEA
Authors: Ghozland Sandy G , Tella Srihari R , Walker Michelle D , Carr Susan M , Sannerud Christine A
Published: June 2021

Pursuant to the Controlled Substances Act (CSA), DEA collects and reviews scientific, medical and other data for substances with abuse potential for possible placement under the CSA. The administrative process for scheduling is ongoing for indiplon, carisoprodol, dextromethorphan, salvinorin A and several petitions requesting a change in the control status of nabilone, methylphenidate product, removal of marijuana and tetrahydrocannabinols (THCs) from schedule I, control of tramadol, control of several substances as schedule III anabolic steroids, decontrol of sibutramine. Numerous hallucinogenic substances have become popular among drug abusers in recent years. DEA is currently reviewing the data for several hallucinogens including 5‐MeO‐DMT, 5‐MeO‐AMT, 5‐MeO‐DET, 5‐MeO‐MIPT, DIPT, 4‐OH‐DIPT, 2C‐I, 2C‐T‐2, and Bromo‐dragonfly for possible control under the CSA. Chemical synthesis/pharmacological studies for DOC, 2C‐C, 2C‐D, and 2C‐E are ongoing to determine if they meet the requirements for possible control under the CSA. To comply with the 1971 Convention on Psychotropic Substances, DEA is reviewing zipeprol, amineptine, mesocarb, 4‐methylthioamphetamine (4‐MTA) and brotizolam, which are not currently controlled or marketed in the United States, for control under the CSA.
Response‐Maintaining Effects of Quinpirole in the Rat: Effects of Operant History and Drug‐Paired Stimuli
Authors: Collins Gregory Thomas , Woods James Henry
Published: June 2021

D2‐like agonists, such as quinpirole (QPRL), maintain responding in rats and monkeys, but are rarely abused in humans. These studies were aimed at examining the influence of operant history and drug‐paired stimuli on QPRL maintained responding in rats. Rats were trained to nosepoke (NP) on a FR1 for cocaine (COC) with a 5 s TO signaled by houselight illumination. Once responding stabilized, COC, QPRL, or saline (SAL) were substituted (SUB) with and without the COC‐paired stimuli, or on a previously inactive lever (LV). During LV SUBs, LV presses produced an injection followed by a 5 s TO signaled by a flashing houselight; NPs still produced the COC‐paired stimuli, but no injection. COC maintained stable responding regardless of whether stimuli were presented, whereas QPRL maintained responding only when the stimuli were paired with injection. During the LV SUB, responding was reallocated from the NP to the LV when COC was available, whereas NPs persisted, and LV presses were low when QPRL was available. These results suggest that the response‐maintaining effects of QPRL are primarily mediated by an enhancement of the conditioned reinforcing effects of the stimuli that were previously paired with COC.

Supported by NIDA grants DA 020669 and F013771
Acamprosate attentuates cue‐induced reinstatement of nicotine seeking behavior in rats
Authors: Manalo Charlene M. , Lacayo Liliana M , Bholat Yasmin , Spivak Inna E , Pechnick Robert N
Published: June 2021

Acamprosate is a glutamate modulator that has been used in the treatment of alcoholism. This investigation tested the hypothesis that acamprosate can inhibit cue‐induced relapse to nicotine self‐administration (SA) in the rat. Rats were trained to lever press for food under a FR5 schedule, then implanted with jugular catheters. After recovery, daily nicotine SA sessions (0.03 mg/kg/infusion) were conducted under a FR1 schedule (days 1‐5), FR2 schedule (days 6‐8) and then a FR5 schedule (days 9‐30 days). Delivery of nicotine was accompanied by the illumination of a cue light directly above the active lever. Next, extinction sessions were run for 14 days, during which responses on the active lever did not result in the infusion of nicotine or the illumination of the cue light. Beginning on the following day, rats received twice daily injections of saline or acamprosate (50, 100 or 200 mg/kg/i.p.) for 7 days. On the 7th day, the response to the previously conditioned cue was tested. The rats were pretreated with acamprosate and the conditions were the same as nicotine SA, but only saline infusions were delivered. Pretreatment with acamprosate attenuated cue‐induced nicotine seeking behavior. These findings suggest that acamprosate might be efficacious in reducing nicotine seeking behavior.
Role of insulin and hyperglycemia in angiogenic sprouting and microvessel survival
Authors: Krishnan Laxminarayanan , Chang Carlos C , Williams Stuart K , Hoying James B
Published: June 2021

Understanding the effects of hyperglycemia on neovascular remodeling is critical especially considering the associated risk of heart disease, non‐healing wounds, and vascular dysfunction. Hyperglycemia has significant, and often contradictory, effects on endothelial cell activities related to neovascularization like endothelial cell proliferation, migration and tube formation. Therefore, it is unclear if the impairment of neovascularization due to hyperglycemia in vivo, involves altered angiogenic sprouting or possibly later post‐angiogenesis maturation and remodeling events. We examined early angiogenic events in a microvascular construct‐based in vitro angiogenesis model over a range of glucose concentrations. Acute hyperglycemia over 5 days did not alter the initial sprout formation. Interestingly, the absence of insulin caused a reduction in microvessel survival and sprouting. This reduction could be partially reversed by addition of IGF but not by DMAQB1, a metabolic insulin analogue. This study demonstrates the resistance of initial microvascular sprouting to acute hyperglycemia and highlights the growth factor like role for insulin in addition to its metabolic effects in neovessel formation. The role of chronic hyperglycemia and matrix glycation must also be examined to fully understand the role of diabetes on microvascular remodeling. Funded by NIH R01 DK078175, R01 EB077683.
VEGF165b the anti‐angiogenic isoform of VEGF inhibits tumor growth in vivo.
Authors: Hamdollah‐Zadeh Maryam , Harper Steve , Bates David
Published: June 2021

To determine the potential of the anti‐angiogenic isoform,VEGF165b for cancer therapy, we compared the inhibitory effects of recombinant human VEGF165b on mouse model of colon cancer with bevacizumab. 2x106 LS174T human colon carcinoma cells were injected subcutaneously (SC) into the intrascapular dorsal region of nude mice. Tumor‐bearing mice were injected intraperitoneally (IP) with saline or bevacizumab (50 ?g), or SC in the lumbar region with 100 ?g VEGF165b bi‐weekly for 2 weeks. The inhibitory concentration of the IC50 was calculated. Growth of tumors was significantly suppressed by SC VEGF165b injection compared to those treated by saline (p<0.001) or bevacizumab (p<0.001), which also significantly reduced tumor growth (p<0001, 2 way ANOVA). SC injection of VEGF165b resulted in a significant reduction of tumor volume from 254±51mm3 to 35.4±17mm3 after injection (p<0.01 compared with before treatment, rmANOVA, Dunnets), whereas bevacizumab stabilised tumor growth (231±22 before compared with 342.± 117mm3, NS). Tumors in saline treated mice grew significantly from 149±46 to 853±362mm3, (p<0.001). The IC50 of VEGF165b was calculated to be 48µg. Administration of VEGF165b inhibits the growth of colon cancer in vivo and indicates that systemic therapy of VEGF165b may be an effective anti‐VEGF tool for the treatment of colon cancer. This work supported by AICR and PhiloGene Inc.
The Auto‐Digestive Hypothesis: A New Mechanism For Development of Leptin Resistance And Obesity
Authors: Mazor Rafi , Schmid‐Schonbein Geert W
Published: June 2021

Obesity is a major health problem and leads to increased morbidity and mortality from cardiovascular complications. Leptin acts on the brain to regulate food intake through its interactions with its receptor (ObR). Defects in leptin production and in ObR leads to the development of obesity, however, obesity is associated with high circulating leptin levels concomitantly with leptin resistance. The mechanism leading to leptin resistance is unknown. We hypothesize that calorie over consumption can lead to increased proteolytic activity in the brain promoting cleavage of key receptors governing food intake. Wister rats were fed high fat diet (HF) for 10 weeks. A control group was provided with regular chow (NC). At 10 weeks, rats were sacrificed and the brain was removed for determination of proteolytic activity and levels of the extracellular domain of the ObR. HF rats had significantly higher body weight compared to NC. Zymography showed increased proteolytic activity in HF rat's brain, which was accompanied with reduced levels of the extracellular domain of the ObR. Our results also suggest MMP‐9 as a possible mediator of ObR cleavage. We conclude that calorie over‐consumption can promote proteolytic activity and cleavage of key receptors in the brain; therefore can mediate the development of obesity. The origin of the proteolytic activity in HF rat's brain remains to be elucidated.
Late‐Stage Captopril Treatment Reduces Atherosclerotic Lesion Progression in Apolipoprotein E Knockout Mice
Authors: Procknow Jesse David , Sprague Randy , Stephenson Alan H.
Published: June 2021

C57BL6 mice in which the gene for apolipoprotein E is deleted (apoE KO) develop significant lesions in the area of the proximal aorta when fed a high fat diet. It was previously reported that apoE KO mice treated with captopril, an angiotensin converting enzyme (ACE) inhibitor, had reduced late stage atherosclerotic lesions after 12 weeks when compared to untreated mice. We wished to determine the effect of captopril on atherosclerotic lesions in a similar model using a shorter timeframe to more closely represent ACE inhibitor use in humans. 6‐week old apoE KO mice were fed a high‐fat (Western) diet for 14 weeks with captopril (25 mg/kg/day) in their drinking water for weeks 12‐14. At 20‐weeks of age, mice were sacrificed and blood was collected via heart puncture. Aortas were fixed and stained with oil red O to visualize lesions. Serum cholesterol levels were higher in Western‐fed apoE KO mice (males 843.41 ± 40.72, females 991.63 ± 85.57 mg/dl) compared to chow‐fed wild‐type mice (control) (males 132.07 ± 4.50, females 95.60 ± 10.74 mg/dl, both p<0.01). Lesions were greater in Western‐fed apoE KO mice versus control mice (males 20.50 ± 0.82 vs 3.38 ± 0.19%, females 20.57 ± 1.12 vs 3.29 ± 0.15%; both p<0.01), and were reduced in male (59.9%, p<0.01) and female (50.1%, p<0.01) captopril‐treated mice. We conclude that two weeks of captopril treatment significantly reduces late‐stage atherosclerotic lesions in apoE knockout mice.
Structural and Functional Heterogeneity in Vascular Remodeling
Authors: Basu Paulomi , Sen Utpal , Kundu Soumi , Tyagi Suresh C.
Published: June 2021

Cardiovascular diseases, such as atherosclerosis is one of the main causes of hypertension, myocardial infarction and heart failure. Although the extent of atherosclerosis is different in different blood vessels, the underlying cause of these differences is not well studied. We tested the hypothesis that gradient blood pressure in different arteries causes structural and functional heterogeneity that drives susceptibility of the arteries to develop varied degree of atherosclerosis. To test our hypothesis we isolated the dorsal aorta, femoral and carotid artery from male Sprague‐Dawley rat (weight ~300 gms) and performed Western blot to determine the level of MMP‐2, MMP‐9 , MMP‐13 and tissue inhibitors of MMP's (TIMP ‐1, ‐3 & ‐4) expression. Histological tissue sections were used to stain with Messon Trichrome and Van Gieson to determine the level of collagen and elastin. Blood pressure was measured. Our results showed that MMP‐2 and MMP ‐9 expressions were relatively high in carotid and femoral artery compared to dorsal aorta. The expression of TIMP‐3 was detected at higher level in the carotid artery compared to other two vessels. The blood pressure of carotid artery was significantly higher than femoral artery. The results suggest that the structural and functional heterogeneity in different vascular beds relates to differential blood pressure. This, in turn, leads to differential expression of matrix proteins, which might be a cause of differential atherosclerotic plaque formation in different vascular beds.
Identification of microvascular network patterning alterations in young spontaneously hypertensive rats
Authors: Yang Ming , Murfee Walter Lee
Published: June 2021

Microvascular rarefaction, defined by a loss of arterioles and/or capillaries, is a common characteristic of hypertension. Recent work in our laboratory suggests that microvascular rarefaction in adult spontaneous hypertension rats is more complex and is also associated with network patterning alterations. The objective of this study was to compare the microvascular architectures in young hypertensive versus normotensive rats. Mesenteric tissues from age‐matched (7‐8 weeks) male spontaneously hypertensive (SHR; n=13), Wistar‐Kyoto (WKY; n=7), and Wistar (n=8) rats were harvested and immunolabeled with PECAM, an endothelial cell marker. SHR microvascular networks displayed reduced branching, increased arteriole/venous shunts and increased arteriole/venous anastomoses proximal to the capillary level. These observations indicate that network patterning alterations in addition to fewer microvessels are present prior to the establishment of hypertension.
NAD(P)H oxidase and reduced nitric oxide bioavailability mediate cytomegalovirus‐induced arteriolar dysfunction
Authors: Stokes Karen Y. , Johnson Eunice , Hamric Candiss , Leskov Igor L.
Published: June 2021

Cytomegalovirus (CMV) has been implicated in several inflammatory diseases, including cardiovascular disease. In cultured endothelial cells CMV promotes oxidative stress and reduces nitric oxide (NO) bioavailability. These responses are some of the earliest vascular responses to cardiovascular risk factors therefore this may be a link between CMV infection and cardiovascular disease. We assessed the roles of NO and NAD(P)H oxidase in arteriolar responses to CMV in vivo. Wildtype (WT), endothelial NO synthase overexpressing mice (eNOS TgN) or gp91phox knockout (gp91ko) mice received mock‐inoculum or 3X104 PFU murine CMV (mCMV) IP. At 9wk post‐infection, intravital microscopy was used to measure endothelium‐dependent vasodilation responses. Vasodilation to acetylcholine (but not sodium nitroprusside or papaverine) was attentuated in mCMV vs mock WT. mCMV‐infected eNOS TgN and gp91ko‐mCMV mice were protected against the impaired arteriolar responses. However in bone marrow chimeric mice where gp91phox was absent on blood cells, but expressed in the vessel wall, mCMV led to impaired vasodilation responses. These data implicate both reduced NO bioavailability and activation of vessel wall NAD(P)H oxidase as mediators of CMV‐induced arteriolar dysfunction. These findings may explain the mechanisms through which CMV contributes to inflammatory diseases. (AHA 0565285B & 0735354N).
Blubber deposition during ontogeny in free‐ranging bottlenose dolphins (Tursiops truncatus): balancing disparate roles of insulation and locomotion
Authors: Noren Shawn Ranee , Wells Randall S.
Published: June 2021

Water conducts heat 25 times faster than air creating an extreme environment for mammalian thermoregulation. Blubber is a key component of dolphin thermoregulation but the cost of descent (COD) to overcome blubber's buoyant force while diving could constrain its deposition. This conflict was examined in 1‐12 year‐old bottlenose dolphins in Sarasota Bay, Florida in summer (mean water temperature: 29.7 ± 0.1 °C) and winter (mean water temperature: 19.2 ± 0.4 °C). Surface area to volume ratio decreased significantly with age. Yearlings had significantly thicker blubber than 2‐12 year‐olds during summer, but this difference diminished by winter as blubber deposition in response to colder water temperature was smaller in yearlings (2 mm increase) compared to 2‐12 year‐olds (3‐6 mm increase). This disparity may be explained by the buoyant force of blubber and the associated locomotor costs to overcome buoyancy while diving. During summer, calculated buoyancy was highest in yearlings, 6.24 ± 0.41 N compared to ‐0.98 ± 0.90 N (neutrally buoyant) for 12 year‐olds. Conversely, all dolphins converged upon a similar buoyant force in winter (8.01 ± 0.56 N). As a result, the calculated mass‐specific COD to overcome buoyancy on a 10 m dive was greatest in yearlings. Balancing energetic demands of locomotion and thermoregulation may limit the flexibility of yearlings to adjust blubber in response to fluctuating water temperatures.
Hypobaria enhances oxygen delivery in grasshoppers
Authors: Harrison Jon Fewell , Bruce Aaron M
Published: June 2021

Hypobaric hypoxia (altitude) theoretically poses less of a challenge to oxygen delivery than normobaric hypoxia for animals with significant diffusive exchange in the gas phase, as increases in the diffusion coefficient match decreases in PO2 as barometric pressure decreases. However hypobaria could have separate stressful effects on collapsible structures. Here we tested the effect of hypobaric hypoxia, normobaric hypoxia, and hypobaric normoxia (constant PO2) on respiratory responses of adult American locusts, Schistocerca americana. Hypobaric and normobaric hypoxia stimulated ventilation frequency, limb movements (escape behavior) and CO2 emission rates. Multiple results supported the importance of aerial diffusion in resting gas exchange of grasshoppers. First, animals in hypobaric hypoxia struggled less than those in normobaric hypoxia at equivalent PO2's. Second, ventilation and CO2 emission rates decreased as barometric pressure decreased at constant PO2. These data indicate that gas‐phase diffusion is an important mechanism of gas exchange in these large insects that exhibit prominent abdominal pumping, and that insects are likely to perform better at altitude than predicted by tests performed in normobaric hypoxia. This research was supported by IBN‐9985857 and IBN 0419704 to JFH.
Clock gene plays an important role in time‐dependent change in thermoregulation during cold exposure in fasted mice
Authors: Tokizawa Ken , Uchida Yuki , Nagashima Kei
Published: June 2021

BACKGROUND

Food deprivation decreases body core temperature (Tb) in mice, which shows time‐dependency, greater in the inactive phase and smaller in the active phase. The purpose of the present study was to clarify the mechanism involved in the change of Tb rhythm during fasting.

METHODS

Male ICR wild‐type (WT) and Clock mutant (CM) mice were housed at 27°C in a 12:12‐h light‐dark cycle. After 48‐h fasting, ambient temperature was maintained at 20°C for each in the light phase (LP) or dark phase (DP). Tb, oxygen consumption, UCP1 mRNA level in the brown adipose tissues, and Fos‐IR cells in the hypothalamus were assessed.

RESULTS

Tb decreased in the cold in all the groups. In WT, the reduction was greater in LP than in DP; however, the reduction was similar in both the phases in CM. Oxygen consumption and UCP1 mRNA increased in the cold in DP of WT and both the phases of CM, whereas remained unchanged in LP of WT. After cold exposure, counts of Fos‐IR cells increased in the medial preoptic area and paraventricular nucleus in all the groups, which were greater in DP of WT and both phases of CM than in LP of WT.

CONCLUSION

These results indicate that fasting attenuates thermoregulatory responses to the cold, depending on time of the day. Neuronal activities in the hypothalamus during the cold exposure may be associated with the thermoregulatory response and its time‐dependency, for which Clock gene plays an important role.
Beta‐GPA‐induced changes in mitochondrial biogenesis are associated with a muscle type‐dependent decrease in RIP140
Authors: Williams Deon , Basaraba Susan , Sutherland Lindsey , Bomhof Marc , Thrush Brianne , Dyck David , Field Catherine , Wright David
Published: June 2021

Beta‐guanidopropionic acid (β‐GPA) is a creatine analogue that leads to reductions in ~P concentrations and increases in markers of mitochondrial content in rodent skeletal muscle. Prior evidence attributes these changes to activation of the 5'AMP pathway; however data suggest RIP140, a negative downstream regulator of PPAR gamma co‐activator 1 alpha (PGC‐1α), is a likely player in the regulation of mitochondrial biogenesis (MB). The aim of this study was to determine if β‐GPA feeding decreases RIP140 protein in skeletal muscle in a fiber type‐specific manner and if it is associated with an increase in mitochondrial content. Male Wistar rats were fed a semi‐purified diet supplemented with (n=12) or without (n=12) 1% β‐GPA for 6 weeks. β‐GPA feeding induced skeletal muscle MB in tricep as shown by increases in CORE1 and COXIV protein and citrate synthase activity. In contrast, no such changes were seen in soleus from β‐GPA‐fed rats. β‐GPA feeding led to a ~75% increase in PGC1α messenger RNA expression in tricep and ~140% increase in soleus. RIP140 protein decreased ~40% in tricep while it was unchanged in the soleus. In summary, β‐GPA feeding led to decreases in RIP140 in rat tricep only, correlating to increases in MB in only this muscle type. This suggests that RIP140 may play a key role in regulating MB downstream of PGC1α and provides a mechanism for fiber type differences in muscle adaptation.

Supported by NSERC
Impact of organic osmolytes on the functional properties of parvalbumin isoforms from a euryhaline stingray
Authors: Heffron Jennifer Kate , Moerland Timothy S
Published: June 2021

Cellular inorganic ion and organic osmolyte concentrations profoundly impact the functional properties of proteins. Here, we focused on the impact of changes in organic osmolyte concentrations on an intracellular muscle protein, parvalbumin (PV), implicated in muscle relaxation. The euryhaline stingray Dasyatis sabina alters both intra‐ and extracellular organic solute concentrations to adjust to the osmotic conditions in its habitat. Two PV isoforms (I and II) present in D. sabina were found to display notably different responses to relevant organic osmolytes‐ betaine, trimethylamine oxide (TMAO), and urea. Dissociation constants (KD) for Ca2+ and Mg2+ ions were determined under physiological conditions and in the presence of each osmolyte. Preliminary data found no significant difference in PVI KD (6.03+ 0.05nm (Ca2+) and 27.55+1.40µm (Mg2+)) under freshwater and marine conditions. PVII (11.09 +0.50nm) was found to possess enhanced calcium binding in the presence of each osmolyte (3.43 + 0.13 nm). A study of varying urea strength revealed PVI lost complete ion binding ability at 3.6M urea. This study may allow insight into how other proteins may be functioning under varying osmotic conditions.
Effect of castration on mouse skeletal muscle oxidative capacity
Authors: Sato Shuichi , White James P , Carson James A
Published: June 2021

Low testosterone is often observed in male patients with cancer, COPD and HIV, and associated with impaired oxidative metabolism in skeletal muscle. Although testosterone has well described effects on muscle mass, less is known about the effect of testosterone on muscle's metabolic capacity. The purpose of this study was to determine if oxidative capacity is reduced in wasting skeletal muscle due to castration and if anabolic steroid (ND) administration can rescue this capacity. 8‐week male C57BL/6 mice were placed into three treatment groups: sham castration (CON, n = 6), castration (CAS, n = 6), and castration with ND administration (n = 6). Gastrocnemius (GAS) muscle succinate dehydrogenase (SDH) activity and PGC‐1 expression were examined. Castrated GAS muscle increased the percentage of low SDH activity fibers (78.2±1.5% vs. 84.5±1.1%, p < .05) and decreased the incidence of high SDH activity fibers (5.0±0.6% vs. 1.3±0.2%, p < .05) compared to CON. ND administration significantly attenuated these changes. PGC‐1 mRNA expression decreased 54% with castration (p = .029), but increased 141% with ND administration (p = .036). These data indicate testosterone availability may regulate oxidative capacity in mouse hindlimb muscle. This study was funded by NIH Grant 5R03AR051434‐02.
Simvastatin represses eukaryotic initiation factor 2B (eIF2B) expression and activity in C2C12 myoblasts with concomitant reductions in protein synthesis
Authors: Tuckow Alexander P , Jefferson Sarah J , Kimball Scot R , Jefferson Leonard S
Published: June 2021

Statins are a widely prescribed class of cholesterol lowering drugs whose use is frequently associated with muscle related ailments. A number of mechanisms have been implicated in statin‐induced myotoxicity including alterations in both protein synthesis and protein degradation. The objective of the current study was to explore the mechanism(s) contributing to the statin‐induced reduction in protein synthesis in a muscle‐derived cell line. C2C12 myoblasts were treated with 10 μM simvastatin or vehicle alone for 24 h in low serum conditions. Cells exposed to simvastatin exhibited reduced rates of protein synthesis as evidenced by [35S] methionine incorporation into protein. The reduction in protein synthesis occurred with a concomitant repression of the guanine nucleotide exchange activity of eIF2B, a regulated and rate‐controlling enzyme known to affect global rates of protein synthesis. Simvastatin treatment also resulted in reduced expression of several subunits of the eIF2B heteropentameric complex at the protein level without alterations in expression of the mRNAs encoding the respective subunits. Finally, increased phosphorylation of the catalytic epsilon subunit at Ser535 was observed ‐ an event consistent with reduced eIF2B activity. These results suggest that repression of eIF2B expression and activity may contribute, at least in part, to the statin‐induced reduction in protein synthesis.

Supported by grant DK15658
Differential Epigenetic Modifications of Histones at the Myosin Heavy Chain Genes in Fast and Slow Skeletal Muscle Fibers and in Response to Muscle Unloading
Authors: Pandorf Clay E , Haddad Fadia , Baldwin Kenneth M
Published: June 2021

In order to delineate the relationship between epigenetic modifications and myosin heavy chain (MHC) gene regulation, we examined histone modifications of the four MHC genes (I, IIa, IIx, IIb) in fast vs. slow fiber‐type rat skeletal muscle, and in a model of muscle unloading, which causes a coordinated shift from slow to fast MHC gene expression in the soleus (sol). We used chromatin immunoprecipitation (ChIP) to examine acetylation of histone H3 (AcH3) and histone H3 lysine 4 trimethylation (H3K4me3). We report that both AcH3 and H3K4me3 correlate with the transcriptional activity, assessed via pre‐ and mRNA, of each of the MHC genes in both fast plantaris and slow sol. Likewise, during dynamic MHC remodeling of sol with unloading, the slow (I, IIa) to fast (IIx, IIb) transition in mRNA parallels alterations in histone modifications (p<0.05). Thus, I and IIa MHC genes become de‐acetylated when their transcription is down‐regulated, while induction of the fast IIx and IIb MHCs occurs in conjunction with enhanced acetylation at those genes. H3K4me3 is similarly altered. These observations demonstrate the feasibility of using the ChIP assay to understand the native chromatin environment in adult skeletal muscle, and also suggest that the transcriptional state of MHC genes are sensitive to histone modifications both in different muscle fiber‐types and in response to altered loading states. Supported by NIH AR30346‐25
Inhibition of myostatin enhances the regenerative potential in atrophied muscle under unloading condition
Authors: Goto Katsumasa , Ohno Yoshitaka , Hashimoto Naohiro , Sugiura Takao , Ohira Yoshinobu , Yoshioka Toshitada
Published: June 2021

It is known that inhibition of myostatin induces muscular hypertrophy. However, effects of inhibition of myostatin on the regenerative potential of atrophied skeletal muscles are still unclear. Present study was performed to examine the effects of inhibition of myostatin on the regenerative potential of atrophied soleus muscles under the unloading condition using the transgenic mice expressing the dominant negative‐expressing form of myostatin (MSTN‐DN). Unloading of soleus was induced by hindlimb suspension (HS). After 2 weeks of HS, the necrosis‐regeneration cycle was initiated by intramuscular injection of cardiotoxin into soleus. Thereafter, HS was maintained for additional 4 weeks. Recovery of muscle weight and protein contents of regenerating muscles was facilitated compared with that of wild type animals. Results from this study indicated that MYSN‐DN has strong stimulating effect on the regenerative potential in muscle under the unloading condition. This study was supported, in part, by Grant‐in‐Aid for Scientific Research (B, 20300218, KG; A, 18200042, TY; S, 19100009, YO) from Japan Society for the Promotion of Science, and Research Grant from KAO Health Science Research (KG).
Regulation of Transcription of Renal Proximal Tubule Na,K‐ATPase by G‐Protein Coupled Receptors
Authors: Taub Mary , Springate James E , Khrovi Sudha
Published: June 2021

Our previous studies indicate that transcription of the Na,K‐ATPase beta subunit gene in MDCK cells is regulated by Prostaglandins, via 2 Prostaglandin Regulatory Elements (PGREs). In order to determine whether 1) our studies with MDCK have a broader physiologic significance, and 2) similar regulation occurs in the renal proximal tubule (RPT), transient transfection studies were conducted with primary rabbit RPT cells. When transfecting using pH ?1‐1141Luc, a beta subunit promoter/luciferase construct, we observed a stimulation by PGE1 and PGE2 , as well as the Gs‐coupled EP2 and EP4 receptor agonists butaprost and PGE1‐OH (respectively). The PGE1 stimulation was inhibited by the antagonists SC51089 and AH6909 (which block Gq coupled EP1 receptors).

Na,K‐ATPase in the renal proximal tubule is regulated by epinephrine and dopamine, which, like PGE, act via G‐ Protein Coupled Receptors (GPCRs). Indeed, 1 µM epinephrine stimulated transcription, as well as the alpha 1 and beta agonists phenylephrine and isoprenaline, respectively. Similarly, dopamine was stimulatory, an effect which was inhibited both by the D1 and D2 receptor antagonists, SCH23390 and L741, 626, respectively. These results are consistent with previously reported chronic stimulatory effects of dopamine on renal Na,K‐ATPase (in addition to acute inhibition), and may be mediated transcriptionally via PGREs.
Does the cytosolic amino ‐terminus of γ‐ENaC function as a nuclear signaling molecule?
Authors: Mironova Elena , Stockand James David
Published: June 2021

Functional ENaC is in the apical plasma membrane of epithelial cells where it's activity is limiting for Na+ (re)absorption and thus, important for electrolyte balance. Enzymes that cleave the channel modify ENaC: including release of the N‐terminal portion of γ‐ENaC. This post‐translational modification increases ENaC activity by increasing channel open probability; however, the role of the section of ENaC cleaved from the core of the channel remains unclear. We previously showed that the amino‐terminal portion of γ‐ENaC when expressed alone targets to the nucleus rather than the plasma membrane. This leads us to wonder whether cleavage of ENaC resulting in release of the N‐terminus of γ‐ENaC results in both an acute activation of the channel and a secondary activation of a nuclear signaling event, similar to Notch signaling and release of the intracellular portion of polycystin. We find that the N‐terminal tail of γ‐ENaC targets to the nucleus decreasing activity of membrane resident channels in a dose‐dependent manner. This is in contrast to the carboxy‐terminal portion of this subunit that does not translocate to the nucleus or affect channel activity. These early results are consistent with the idea that post‐translation modification of ENaC by proteases has two effects: an acute activation of the channel and initiation of a negative‐feedback pathway involving nuclear signaling by the cleaved portion of ENaC.
Upregulation of the thiazide‐sensitive sodium chloride cotransporter in the kidney is associated with the hypertension in D3 dopamine receptor heterozygous (D3 ‐/+) mice
Authors: Wang Xiaoyan , Escano Crisanto S , Asico Laureano , Jones John E , Jose Pedro A
Published: June 2021

D3 dopamine receptor (D3R) knockout (D3‐/‐) mice have hypertension related to an increased protein expression of thiazide‐sensitive sodium chloride cotransporter (NCC) in the renal distal convoluted tubule. D3R heterozygous mice (D3 ‐/+) are also hypertensive. To determine if a mechanism involved in the pathogenesis of hypertension in D3 ‐/+ is similar to that in D3‐/‐ mice, we measured renal NCC protein abundance in D3‐/+ and their wild type (D3+/+) littermates. On normal NaCl intake, blood pressure (BP, telemetry) was higher in D3‐/+ than in D3+/+ mice but body weight, serum electrolytes and urinary sodium excretion were similar. NCC protein expression in D3‐/+ mice was increased in the renal plasma membrane‐enriched (136±7) and intracellular‐vesicle enriched fractions (154±7) (% of D3+/+, n=6/group). To determine the role of the increased NCC expression in the hypertension of D3‐/+ mice, they were treated with hydrochlorothiazide (HTZ, 30 mg/Kg X 3 days, IP), a specific NCC inhibitor. There was a more than 2‐fold increase in HTZ‐induced natriuresis in D3‐/+ (19.2±3.0) relative to D3+/+ mice (8.1±2.3)(nEq/h, 6 hours post HTZ injection,n=5/group). BP decreased sooner (on day 2 in D3‐/+ mice and on day 3 in D3+/+ mice) and to a greater extent in D3‐/+ (21±2) than in D3+/+ mice (14±1)(mm Hg, n=4/group). We conclude that the hypertension of D3R deficient mice is associated with upregulation of the renal NCC.