bluuebunny/crossref_metadata_2025 huggingface dataset:
Multi‐scale Modeling of Collecting Lymphatic Vessels: From Ion Channel Activity to Lymph Transport
Authors: Moshkforoush Arash , Kapela Adam , Tsoukias Nikolaos
Published: November 2023

Exact physical, molecular and cellular mechanisms underlying the transport of the lymph within the lymphatic system is highly complicated, and for the most part is poorly understood. This proposed research outlines our recent effort in developing a multiscale computational model of collecting lymphatic vessel segments (lymphangions), in which the transport function at macroscale level can be analyzed by integrating underlying mechanisms at cellular and molecular levels. This outlined approach can provide a useful platform to study the function or dysfunction of the collecting lymphatic vessels in normal and diseased states, respectively. Detailed mathematical expressions of electrophysiological properties of major ionic channels, membrane currents and intracellular stores present in a single lymphatic smooth muscle cell (LSMC) has been derived from independent experimental studies in literature. The single LSMC model predicts the detailed dynamics of calcium and membrane potential at the whole cell level via integrating individual membrane and intracellular components. A model of the lymphangion wall is constructed via connecting LSMCs through the incorporation of homocellular gap junctions between the neighboring cells. This multicellular model has the potential to lay bare on complex intra‐ and intercellular electrical signaling and propagating ionic waves. The detailed electrophysiological model of the lymphangion wall is combined with a biomechanics and fluid dynamics model to form a mathematical model of a section of the collecting lymphatic vessel (i.e. a series of lymphangions separated by unidirectional valves). This model allows us to study the real‐time relationship between the changes in the diameter and pressure of the vessel segments and changes in the concentration of ionic species. The single LSMC model successfully predicts calcium and membrane current oscillations at physiological amplitude and frequencies without the presence of an agonist. The multicellular model suggests that the chloride current is the major contributor in synchronizing the LSMC spontaneous contractions within one lymphangion, while the presence of pressure‐sensitive channels accounts for anti‐phase contractions observed in neighboring lymphangions. This proposed model is a novel attempt that synergistically takes into account the biomechanics of the lymphatic vessel wall, fluid dynamics, and the electrophysiology of major ionic species in the cells constituting the lymphatic vessels.

Support or Funding Information

Supported by National Institutes of Health grant HL121778
Mitochondria Modulates Calcium Pulsars Kinetics in Native Endothelial Cells from Mouse Mesenteric Arteries
Authors: Toussaint Fanny , Béziau Delphine , Charbel Chimène , Blanchette Alexandre , Mayer Gaétan , Ledoux Jonathan
Published: November 2023

Endothelial control of vascular tone is modulated by local calcium (Ca2+) signaling events like Ca2+ pulsars, an IP3R‐dependant Ca2+ release restricted to myoendothelial projections (MEP). Aside from the recent report of Ca2+ pulsars being modulated by CaMKII, mechanisms regulating endothelial spontaneous Ca2+ release remain unidentified. Mitochondria are important modulators of intracellular Ca2+ dynamics in various cell types and, we hypothesized mitochondria may control endothelial Ca2+ pulsars. High‐speed confocal microscopy was used to investigate mitochondria's influence on endothelial Ca2+ pulsar in cut‐open mouse mesenteric arteries (en face configuration). Preventing mitochondrial Ca2+ uptake by uncoupling with FCCP uncovered a new Ca2+ pulsar site population that had a lower frequency (≈+30%) but larger diffusional spread (+57%) than controls. Inhibition of mitochondrial Ca2+ uptake via the uniporter using Ru360 increased the spread of Ca2+ pulsars (≈+30%), though kinetics were not significantly altered. Significantly, inhibition of mitochondrial Ca2+ export (CGP37157, CsA) increased the number of active Ca2+ pulsar sites, each with a decreased bursting frequency. In summary, these results suggest that mitochondrial uptake and release of Ca2+ acts as a critical local modulator of Ca2+ pulsar kinetics and may therefore be crucial to endothelial control of vascular tone.

Support or Funding Information

FRQS, HSFC, CFI, SQHA and CIHR
Inhibition Of Inward‐Rectifier K+ Channels In Vascular Smooth Muscle Restores Myogenic Tone Development In Mouse Bladder Arterioles
Authors: Tykocki Nathan R. , Heppner Thomas J. , Nelson Mark T.
Published: November 2023

The urinary bladder is highly vascularized, and chronic decreases in blood flow are linked to bladder dysfunction. While increases in intraluminal pressure constrict most arterioles (“myogenic tone”), mouse bladder arterioles do not exhibit a myogenic response to pressure. We examined the electrophysiological and vasoactive properties of mouse bladder feed arterioles to determine why myogenic tone is absent. In isolated pressurized bladder arterioles, smooth muscle cell membrane potential was hyperpolarized at 20 mmHg (−72.8±1.4 mV), and did not change significantly when intraluminal pressure was increased to 80 mmHg (−74.3±2.2 mV). However, increasing extracellular K+ from 5.9 mM to 60 mM caused a significant depolarization (−22.7±0.2 mV) at 80 mmHg, which was accompanied by vessel constriction. Given that inward‐rectifier K+ (KIR) channels can have a strong influence on membrane potential in smooth muscle cells, membrane potential was again measured in the presence or absence of the KIR channel blocker BaCl2 (100 μM). In the absence of BaCl2, smooth muscle cell membrane potential remained hyperpolarized at both 20 to 80 mmHg (−70.4±1.9 mV and −72.2±3.1 mV, respectively). In the presence of BaCl2, membrane potential depolarized significantly at both 20 mmHg (−56.2±3.1 mV) and 80 mmHg (−43.3±2.0 mV). To determine if KIR channel inhibition could restore myogenic tone, diameter of isolated pressurized bladder arterioles was measured at increasing pressures (10–80 mmHg) in the presence and absence of 100 μM BaCl2. BaCl2 had no effect on vessel diameter at 20 and 40 mmHg, but resulted in the development of significant myogenic tone at both 60 and 80 mmHg (25.5±9.1% and 41.5±3.9% constriction, respectively). These data support the concept that, in urinary bladder arterioles, KIR channel conductance cannot be overcome by pressure‐induced depolarizing conductances, and thus smooth muscle cell membrane potential remains hyperpolarized and the arterioles do not constrict as intraluminal pressure increases. This represents a novel adaptation to maintain blood flow into the bladder during filling.

Support or Funding Information

Supported by NIH R37DK053832 and K01DK103840.
PDE6 Isoforms Are Expressed in Native Endothelium but not in Cultured Cell Lines
Authors: Blanchette Alexandre , El Jetlawi Fatma , Toussaint Fanny , Charbel Chimene , Comtois Philippe , Ledoux Jonathan
Published: November 2023

Nitric oxide (NO) is a major component of endothelial regulation of vascular homeostasis.. Diffusion of NO to the underlying vascular smooth muscle cells (VSMCs) will trigger Cyclic GMP (cGMP) production through activation of soluble guanylate cyclase leading to myocyte relaxation. As a second messenger central to numerous signalling pathways, proper control of cGMP level is critical. Although modulation of cGMP levels by phosphodiesterases (PDEs) is well established in smooth muscle through PDE5, few studies have focused in cGMP regulation in endothelial cells. Amongst cGMP‐specific PDEs, PDE5 and PDE9 have been reported in VSMCs and ECs. PDE6, a retina‐restricted cGMP‐specific PDE, has considerable similarities with PDE5, including overlapping. The current study establishes the expression of PDE6 isoforms in endothelial cells and their impact on local cGMP levels. Interestingly, all PDE6 isoforms (α, β and γ) were found in endothelial cell from mouse mesenteric arteries. Complementary approaches provide compelling evidences of the expression of PDE6 isoforms at mRNA and protein levels in native endothelial cells. However, PDE6a and b were not detected in endothelial cell lines. Subcellular distribution of PDE6 and 5 was sought using confocal microscopy. Whilst PDE5‐ and PDE6γ‐associated immunofluorescence was homogeneously distributed, PDE6α and PDE6β appeared preferentially localized within the perinuclear region. Mathematical modeling demonstrated the influence of PDE localization on local cGMP concentrations. Indeed, subcellular endothelial cGMP level following a NO pulse is unsymmetrical, with the peri‐nuclear region showing very little increase in cGMP. These results strongly suggest that PDEs, especially PDE6α and PDE6β, might have an significant impact on cGMP‐mediated cellular pathways in native endothelium through the fine‐tuning of cGMP microdomains.

Support or Funding Information

Supported by FICM
Lipid composition of the myoendothelial junction allows for compartmentalized eNOS function
Authors: Biwer Lauren , Taddeo Evan , Hoehn Kyle , Straub Adam , Isakson Brant
Published: November 2023

Cell communication within the wall of resistance arteries is a key contributor to vascular tone. In resistance arteries that are responsible for blood pressure regulation, the endothelial cell (EC) can project through the internal elastic lamina and make contact with the neighboring smooth muscle cell (SMC), forming a myoendothelial junction (MEJ). Our lab has previously shown that endothelial nitric oxide synthase (eNOS) is present in the apical EC and the basal EC (i.e, the MEJ). To test if the apical and MEJ pools of eNOS acted in sync or separately, we stimulated an in vitro co‐culture of human SMC and EC with 50uM phenylephrine (PE). This increases MEJ eNOS phosphorylation (eNOS‐P) at S1177 but not in the rest of the EC. Conversely, 10uM bradykinin (BK) increases EC eNOS‐P but does not affect MEJ eNOS‐P. Inhibiting gap junctions abrogates the MEJ eNOS‐P seen after PE, but has no effect on BK stimulated eNOS‐P. We hypothesized that differential lipid composition between the apical EC and MEJ may account for the differential localization and function of eNOS. What we found is that the lipid composition of co‐cultured EC & SMC is unique and suggests protein kinase C (PKC) localization at the MEJ. Indeed, PKC activity is significantly higher at the MEJ than the rest of the EC. Because PKC activity also relies on ER calcium release, we used the inhibitors thapsigargin and xestospongin C which caused significant decreases in MEJ eNOS‐P after PE. Furthermore, pharmacological inhibition of PKC significantly decreases MEJ eNOS‐P. To test for a functional effect, application of tumor necrosis factor alpha significantly increases leukocyte adhesion. When BK was added, this significantly decreased the percentage of adherent calcein green loaded THP‐1 monocytes while PE had no effect. Conversely, cGMP levels increase 5 minutes after PE and this increase is abrogated with gap junction inhibition. Underscoring the role of the MEJ in this pathway, SMC grown in flasks (without the presence of EC and thus, no MEJ formation) do not increase cGMP production after PE. Thus, we hypothesize that the unique, local lipid composition of the MEJ facilitates the greater PKC activity and distinct activation and function of MEJ eNOS versus the apical eNOS.

Support or Funding Information

American Heart Association National Institute of Health
Limb specific differences in vasomotion response to varying rates of local heating
Authors: Del Pozzi Andrew Thomas , Miller James T , Hodges Gary J
Published: November 2023

We examined skin blood flow (SkBF) and vasomotion in the forearm and leg using laser‐Doppler fluxmetry (LDF) and spectral analysis to investigate endothelial (0.0095 – 0.021 Hz), sympathetic (0.021 – 0.052 Hz), and myogenic (0.052 – 0.145 Hz) activity in response to slow (0.1°C·10 s−1) and fast (0.5°C·10 s−1) local heating. Based on previous investigations we hypothesized that both limbs would respond with higher initial and sustained vasodilator activity in response to the fast‐rate of heating. Secondly, we hypothesized that the vasomotion responses would differ between the forearm (greater sympathetic activity) and the leg (greater endothelial activity). At 33°C (thermonuetral) endothelial activity was higher in the legs than the forearms (P ≤ 0.02). Fast‐heating increased SkBF more than slow heating in both the forearm (169 ± 30 mV vs. 151 ± 28 mV; P = 0.037) and the leg (159 ± 32 mV vs. 130 ± 12 mV; P = 0.002). At onset of 42°C, endothelial activity was increased in both regions during the fast‐heating protocol (P = 0.009 forearm; P = 0.02 leg). There was greater sympathetic activity observed in the forearm (7.8825 mV2/Hz) than the leg (2.5 mV2/Hz) during the first 5 min of the fast‐heating (P = 0.004) protocol. With prolonged heating (42°C for 35 min), endothelial activity was higher following fast heating compared to slow heating in both the forearm (11.855 vs. 4.828 mV2/Hz; P = 0.01) and leg (9.338 vs. 4.319 mV2/Hz; P = 0.011). In the skin of the forearm compared to the leg, endothelial (11.855 vs. 9.338 mV2/Hz; P = 0.047), myogenic (1.6055 vs. 1.2305 mV2/Hz; P = 0.049), and sympathetic (9.126 vs. 5.526 mV2/Hz; P = 0.046) activity were higher for the fast heating protocol. These results confirm regional differences in the response to local heating and suggest that the increase in SkBF in response to rapid local heating at both the forearm and leg sites is due to increased endothelial and sympathetic activity.

Support or Funding Information

none
Ossified Bone Marrow Blood Vessels, Trabecular and Cortical Bone Properties and Hematological Parameters in 6‐ and 18‐month‐old Fischer 344 Rats
Authors: Guderian Sophie , Lee Seungyong , McLane Mary Ann , Prisby Rhonda
Published: November 2023

BACKGROUND

We previously discovered ossification of bone marrow blood vessels (BMBV) in 6‐ and 24‐month‐old F‐344 rats, whereby some BMBV had converted into bone (Prisby, 2014). Recently, we described the presence of ossified BMBV as early as 1 month in F‐344 rats, suggesting that this vascular pathology begins early in life (Guderian, ASBMR 2015). Since bone marrow is the site for blood cell production and blood cells access the peripheral circulation via blood vessels, we sought to assess whether BMBV ossification coincides with declines in red and white blood cell count. We examined BMBV ossification, trabecular and cortical bone properties, and hematological parameters as a function of advancing age.

METHODS

Left femora and whole blood samples were collected from young (6 mon; n=5) and aged (18 mon; n=5) male Fischer‐344 rats. Bone microarchitecture (BV/TV, %; Tb.N,/mm2; Tb.Th, mm & Tb.Sp, mm) and trabecular bone density (g/cm3) in the distal femora and ossified vessel volume (OsVV) and ossified vessel density (g/cm3) in femoral shafts were assessed by mCT (Scanco Medical). Cortical bone parameters (cortical thickness, μm; cortical bone density, g/cm3 & cross sectional moment of inertia [CSMI, mm4]) were also compared between groups. SMI was compared between trabecular bone and ossified BMBV. Complete blood counts were determined with a Coulter ® Ac•T diffTM Analyzer (Beckman).

RESULTS

Body mass (p<0.05) increased with age. Trabecular BV/TV tended to be lower (p=0.056) in young (20.5±1.2%) vs. aged (25.0±1.5%) rats, resulting from decreased (p<0.05) Tb.Th (young, 68.84±2.14 mm vs. old, 89.12±2.65 mm). OsVV was similar between young (0.033±0.0120%) and aged (0.040±0.006) rats. SMI values for OsVV (young, 3.5±0.1 and old, 3.8±0.1) were indicative of cylindrical vs. the plate‐like shapes of trabecular bone (young, 1.8±0.1 and aged 0.8±0.1). Trabecular (young, 908.8±3.6 g/cm3 vs. aged, 935.2±3.6 g/cm3) and cortical (young, 1184.1±5.9 g/cm3 vs. aged, 1237.6±7.8 g/cm3) bone was denser with age (p<0.05). Trabecular bone density was similar to ossified vessel density (young, 882.1±18.9 g/cm3 vs. aged, 923.4±4.7 g/cm3). Cortical bone thickness (young, 724±3 μm vs. aged, 805±26 μm) and CSMI (young, 13.8±0.9 mm4 vs. aged, 22.2±1.8 mm4) were increased (p<0.05) with age. The complete blood count variables (e.g., WBC, RBC, Hgb, Hct, etc.) did not differ between groups.

CONCLUSIONS

OsVV and hematological parameters did not differ between 6‐ and 18‐month old rats. However, trabecular BV/TV, trabecular and cortical bone density, cortical bone thickness and CSMI were higher in 18‐month‐old animals.

Support or Funding Information

Supported by National Institutes of General Medical Sciences (P20 GM103446; Project PI: R. Prisby)
A splice variant of smooth muscle myosin phosphatase regulatory subunit tunes arterial reactivity and suppresses response to salt loading
Authors: Reho John , Kenchegowda Doreswamy , Asico Laureano D. , Fisher Steven
Published: November 2023

Nitric oxide (NO) and reactive oxygen species (ROS) through activation of cGMP‐dependent protein kinase (cGK1a) activate smooth muscle Myosin Phosphatase (MP), thereby causing de‐phosphorylation of myosin and vasorelaxation. cGK1a is targeted to the MP regulatory subunit (Mypt1) via Leucine Zipper (LZ)‐mediated hetero‐dimerization. Isoforms of Mypt1 with variable presence of the C‐terminal LZ motif are generated by alternative splicing of the 31 nt Exon24 (E24). We have demonstrated that the expression of these isoforms is vessel‐specific, developmentally regulated and modulates in disease. Based on correlations of expression and function and biochemical assays we have proposed a model in which Mypt1 E24 splicing tunes arterial sensitivity to NO/ROS mediated vasorelaxation. To directly test this model LoxP sites were inserted in introns flanking E24 and bred into smMHCCreER for Tamoxifen‐inducible and smooth muscle‐specific deletion of E24 from Mypt1 (E24 cKO). This converts Mypt1 to the E24‐ splice variant coding for the LZ+ isoform. We generated an allelic series of mice: Cre+ with 0, 1 or 2 floxed alleles and measured blood pressure (BP) by telemetry and arterial function by wire myography. E24 cKO had significantly lower systemic BP with no difference between hetero‐ and homo‐zygotes. There was no effect on the circadian variation in BP. Vasorelaxation of 1st order mesenteric arteries (MA1) to the NO donor DEA/NO, and its 2nd second messenger cGMP in permeabilized and calcium clamped arteries, was markedly enhanced again with no difference between hetero‐ and homo‐zygotes. The only parameter in which these mice differed was a marked suppression of force generation to the a‐adrenergic agonist phenylephrine (PE) in the homozygotes. This was specific to this signaling pathway as there were no changes in force generation to the thromboxane analogue U46619, Angiotensin II, KCl depolarization or calcium. Two weeks of salt loading (4% sodium diet) increased MA1 force generation to PE in wild type mice. This response was abrogated y in the homozygous Mypt1 E24 cKO mice. We conclude that Mypt1 E24 splice variants tune arterial sensitivity to vasodilators such as NO/ROS signaling though cGK1 activation of MP. Targeting of therapies to the contractile apparatus of microvascular smooth muscle may have salutary effects in the treatment of hypertension and other cardiovascular diseases in which increased vascular resistance has a dominant role.

Support or Funding Information

NIH R01‐HL066171, 7R37‐HL023081, T32‐HL072751
Spectral analysis measured in cooling‐evoked hemodynamic perturbations of rats indicates endogenous nitric oxide facilitating sympathomyogenic oscillations
Authors: Tung Che‐Se , Liu Yia‐Ping , Lin Yi‐Hsien , Lin Yu‐Chieh , Lee Po‐Lei
Published: November 2023

Rapid cooling stress (CS) elicits hemodynamic perturbations (CEHP). We previously reported that CEHP is highly relevant to the sympathetic activation and nitric oxide (NO) production. This study identifies the role of NO in CEHP. Conscious rats were pretreated with the NO‐synthase inhibitor (L‐NAME) alone or superimposition of sympatholytic interventions using hexamethonium (HEX) or guanethidine (GUA), and then subjected to a CS trial. Cardiovascular indices were monitored via an implanted telemetric device throughout experimental protocol. The analyses included measurements of blood pressure (BP); heart rate (HR); the variability of BP and HR (BPV, HRV); spectral powers and coherence linkage at very‐low, low, and high frequency regions (VLF: 0.02–0.2 Hz, LF: 0.2–0.6 Hz, and HF: 0.6–3.0 Hz); and dicrotic notch (Dn). When compared with control vehicle under CS, the L‐NAME alone (a) increased BP, Dn, VLFBPV, VLFHRV, LFHRV, and HFHRV but decreased HR, HFBPV, and LF/HF ratio; (b) converted negative correlation into positive correlation for LF pair (LFHRV/LFBPV) and VLF pair (VLFHRV/VLFBPV); and (c) enhanced the coherence linkage between BPV and HRV at the LF region but weakened them at the HF region. We also noted divergent patterns of spectra responsiveness to HEX or GUA superimposed on L‐NAME. The effects of HEX+L‐NAME versus GUA+L‐NAME, particularly on LF and VLF between BPV and HRV, indicated an essential role for a functional adrenal medulla to release epinephrine under CS. Our results indicate that endogenous NO play an inhibitory role on the sympathovascular activation and subsequently the myogenic vascular oscillations in CEHP.

Support or Funding Information

Grant in aid: the Ministry of Science and Technology (MOST 102 &103‐2320‐B‐350‐001) and the National Defense Medical Center (MAB101‐40; MAB‐102‐81), Taiwan, R.O.C.
Relationship between the elevated orexin sensitivity via OX2R in RVLM and an increased arterial pressure in rats fed a high fat diet
Authors: Yamaguchi Aoi , Obata Koji , Morita Hironobu
Published: November 2023

It is known that metabolic syndrome is a risk factor of myocardial infarction and cerebrovascular disorder. Rats fed a high fat diet have been used as an animal model of metabolic syndrome, i.e., increases in body mass, arterial pressure (AP), and leptin in plasma. We hypothesized that sympathoexcitation caused by the elevated orexin sensitivity in rostral ventrolateral medulla (RVLM) induces an increased AP in HFD. We employed Sprague Dawley rats fed a high fat diet (HFD) or a normal diet (ND) for 10 weeks from 3 weeks old. We measured orexin A in cerebrospinal fluid by ELISA and orexin receptors (OX1R, OX2R) mRNA expression in RVLM by RT‐PCR. There was no significant difference in orexin A between HFD and ND (HFD: 62.5 ± 2.1 pg/mL, ND: 58.1 ± 3.0 pg/mL). On the other hand, OX2R mRNA in HFD was significantly higher than that in ND (HFD: 0.98 ± 0.21, ND: 0.50 ± 0.10). To test the hypothesis, we injected orexin B (175 pmol in 50 nL) to RVLM under urethane + α‐chloralose anesthesia with measuring AP. Orexin B induced a larger AP response in HFD than ND (area under the curve in HFD: 1675 ± 318 mmHg·180 s, ND: 814 ± 255 mmHg·180 s). We also injected OX2R antagonist, TCS‐OX2‐29 (20 nmol in 2 μL) to the lateral ventricle. Although OX2R antagonist had no effect on AP in ND, it decreased AP in HFD (area under the curve in HFD: − 509 ± 113 mmHg·180 s, ND: 171 ± 50 mmHg·180 s). These results suggested that the elevated orexin sensitivity in RVLM induces a chronic increased AP in HFD. To further examine this, whether AP in HFD is decreased by chronic injection of OX2R antagonist has to be determined in a future study.
Over‐Expression of Osteopontin Significantly Increases Cardiomyoycte Size
Authors: McBride Martin , Downie Laura , Martin Tamara , McClure John D , Nicklin Stuart , Graham Delyth
Published: November 2023

Introduction

The stroke‐prone spontaneously hypertensive rat (SHRSP) is an excellent model of human cardiovascular disease and develops increased left ventricular mass index (LVMI) in young animals, prior to the onset of hypertension. We identified a quantitative trait locus (QTL) for LVMI on chromosome 14. Using a combination of chromosome 14 congenic strains and heart gene expression profiling, we identified positional candidate genes within the chromosome 14 congenic interval, including osteopontin. The aim of this study was to validate osteopontin as a functional candidate gene affecting cardiomyocyte size.

Methods

H9c2 myoblast cells were transfected with osteopontin cloned into pcDNA1 (3ug), using Xfect transfection reagent. Cells were seeded 24hrs prior to transfection at a seeding density of 3.4×104 cells/well, conditioned media was obtained from cells post‐transfection and incubated with fresh H9c2 cells for 48hrs. EVOS (crystal violet) microscopy images were analysed using ImageJ software. Cell data was analysed using ANOVA with Dunnett's post hoc test.

Results

Significantly increased osteopontin mRNA expression was confirmed in transfected cells by qRT‐PCR (RQ cells 1.0±0.09, pcDNA1 2.0±0.31, SHRSP osteopontin cDNA 58.2±4.55*, *p<0.001 compared to cells only). Osteopontin transfected cells were significantly increased compared to cell and pcDNA1 transfection controls (cells 71.4±3.4, X‐fect 74.0±1.4, pcDNA1 81.4±2.1*, SHRSP osteopontin cDNA 138.1±3.1*, *p<0.001 compared to cell control). Conditioned media from each group was prepared and added to cells with a significant increase in osteopontin transfected cells, compared to controls (cells 67.3±1.1, X‐fect 65.5±0.8, pcDNA1 67.9±1.1, SHRSP osteopontin cDNA 145.3±3.0*, *p<0.001 compared to cell control).

Conclusion

This data confirms that over‐expression of osteopontin increases cell size in the H9c2 model. This confirms osteopontin as a positional and functional candidate gene underlying the left ventricular mass index QTL on chromosome 14 in the SHRSP rat.

Support or Funding Information

British Heart Foundation and the BBSRC
Propofol dialates coronary arteries in Vitro via Endothelial TRPA1 and TRPV1 Channels
Authors: Roy Pritam Sinha , Bratz Ian , Damron Derek
Published: November 2023

Introduction

Transient receptor potential (TRP) ion channels have emerged as key regulators of vasomotor tone. Propofol is an intravenous anesthetic with numerous benefits, but also associated with side effects such as hypotension and acute pain upon infusion. Our objective is to determine the role of TRPA1 and TRPV1 ion channels on mediating propofol‐induced vasodilation of mice coronary arteries in vitro.

Methods

Hearts were excised from anesthetized mice and coronary arterioles were dissected from control C57Bl/6J, TRPA1−/−, TRPV1−/− and double‐knockout mice (TRPAV−/−). Isolated microvessels were cannulated and secured in a temperature‐controlled chamber and allowed to equilibrate for 1 hr. Vasoreactivity studies were performed in pre‐constricted microvessels to assess the effect of propofol. Also, immunoblot analysis and immunostaining were performed on mice coronary artery endothelial cells (MCAECs). Intracellular Ca(2+) concentration was measured in individual MCAECs via fluorescence microscopy.

Results

In vitro, propofol‐induced relaxation was unaffected in TRPV1−/− vessels, markedly attenuated in TRPA1−/− vessels and abolished in TRPAV−/− vessels. Immunoblot and immunocytochemical analysis detected TRPA1 and TRPV1 channel proteins in MCAECs. Moreover, single application of propofol and capsaicin caused an increase in intracellular [Ca]2+ in MCAECs, that was abolished in presence of the TRPA1 antagonist, HC‐030031 and and TRPV1 antagonist capsazepine.

Discussion

Our coronary in vitro data indicate that TRPA1 is directly involved whereas TRPV1 appears to be indirectly involved because deletion of TRPV1 alone has no observable effect on propofol‐induced depressor response.

Support or Funding Information

HL‐65701 from the NationalHeart, Lung and Blood Institute, Bethesda, Maryland.
SERCA inhibition attenuates medial thickening in an organ culture model of coronary artery disease
Authors: Rodenbeck Stacey Dineen , Barnard April L , Sturek Michael
Published: November 2023

Atherosclerotic coronary artery disease (CAD) involves de‐differentiation of coronary smooth muscle (CSM) from a quiescent, contractile phenotype to a proliferative, migratory phenotype. We tested the hypothesis that sarco‐endoplasmic reticulum Ca2+ ATPase (SERCA) function is causal in CSM transition from contractile to proliferative. We measured proliferation with immunohistochemical analysis of Ki‐67 expression in both mild and severe atherosclerotic plaques. Ki‐67 expression peaks early in CAD progression, dropping off in severe CAD, likely because CSM either further differentiate into osteogenic CSM or become apoptotic. We concurrently examined SERCA function with fura‐2 assessment of caffeine‐induced sarcoplasmic reticulum (SR) Ca2+ store release and subsequent recovery. SERCA function is elevated in mild, early CAD and decreases in more advanced, severe CAD, mirroring Ki‐67 expression patterns. We cultured coronary arterial segments in DMEM with 180 mg/dL glucose and 20% fetal bovine serum for fourteen days, in the presence and absence of the selective SERCA inhibitor, cyclopiazonic acid (CPA). Initial results indicated that CPA treatment completely prevented culture‐induced medial thickening as assessed by histological staining. CSM were enzymatically dispersed and loaded with fura‐2/AM. CPA was absent from the dispersal and experimental solutions. SR Ca2+ store release and subsequent recovery below baseline levels were increased in CSM from CPA‐treated arteries, suggesting that functional inhibition induces of SERCA expression. We will examine SERCA and Ki‐67 expression in future studies to further examine the role of SERCA in the contractile‐to‐proliferative transition in atherosclerotic CAD.

Support or Funding Information

NIH HL062552, IUSM CECARE, AHA 15PRE25280001
CTRP3 Prevents ETOH‐ Induced Hepatocyte Apoptosis
Authors: Dunlay Samantha , Peterson Jonathan M
Published: November 2023

Alcohol abuse is the third leading cause of preventable death in the United States. Our lab works with the novel protein C1q TNF Related Protein (CTRP3), which is secreted from adipose tissue and circulates in the blood stream. Our past lab experiments show that CTRP3 prevents nonalcoholic fatty alcoholic liver disease. Nevertheless, the effects of CTRP3 on alcoholic fatty liver disease (AFLD) are still unknown. Our lab to date, has determined that CTRP3 prevents alcohol‐induce hepatocyte (liver cell) death. The purpose for this project is to understand how. Previous research has demonstrated that ethanol consumption increases apoptosis (programmed cell death) in hepatocytes through an inappropriate elevation in apoptotic signaling. Our working hypothesis is that CTRP3 prevents ethanol‐induced hepatocyte cells death by suppressing ethanol‐induced elevations in pro‐apoptotic signaling. To test our hypothesis we examine the effects of ethanol plus/minus CTRP3 treatment on the Bcl‐2 family of apoptotic regulatory proteins. The Bcl‐2 family can either induce (pro‐apoptotic) cell death or inhibit cell death (anti‐apoptotic).

Methods

Hepatocytes were treated with ethanol (100 mM) plus/minus recombinant CTRP3 and Bcl‐2 family protein content was determined through western blot analysis. We measured the specific apoptotic regulator proteins: Bcl‐2‐associated X protein (BAX), which is pro‐apoptotic, and B‐cell lymphoma 2 (BCL‐2) and (BCL‐XL), which are anti‐apoptotic. Western blots were performed using standard techniques; briefly, protein samples are separated by size through SDS‐PAGE (PolyAcrylamide Gel Electrophoresis). The proteins are then transferred to a nitrocellulose membrane and relative protein concentrations are detected by chemiluminescence.

Results

ethanol treatment increases the amount of BAX and decreases the amount of BCL‐XL but not BCL‐2 protein content in the hepatocytes. However, treatment with CTRP3 stopped the ethanol‐induced elevations to BAX and reductions in BCL‐XL. In conclusion this data supports our hypothesis that CTRP3 prevents ethanol‐induced hepatic cell death.

Support or Funding Information

East Tennessee State University RDC: E82262

NIAAA: R03AA023612
Loss of pancreatic Mig6 improves glucose homeostasis during diet‐induced obesity
Authors: Olivos Naomie , Fueger Patrick T
Published: November 2023

In response to nutritional overload, functional beta cell mass normally expands to compensate for diet‐induced insulin resistance, thereby preserving glucose homeostasis. In the pathogenesis of type 2 diabetes (T2D), however, the compensation is insufficient, as beta cell dysfunction and destruction develop, leading to a mismatch between functional beta cell mass and insulin sensitivity. Thus, targeting beta cell proliferation as well as beta cell secretory capacity (i.e., function) and survival could prevent the onset of the disease. We have been investigating the impact of the elevated levels of mitogen inducible gene 6 (Mig6) in beta cells during glucolipotoxicity, as this milieu is thought to contribute to T2D. Mig6 is an adaptor protein that impairs EGFR signaling, a critical cell survival and proliferation pathway. Thus, we hypothesized that ablating Mig6 would fortify functional beta cell mass and preserve glucose homeostasis during diet‐induced obesity and insulin resistance. To this end, we compared mice lacking pancreatic Mig6 (PKO) and their wild‐type (WT) littermates fed either a low fat or high fat diet. No observable phenotypic differences were observed between either genotype fed the low fat diet. High fat feeding increased body mass and decreased glucose tolerance in WT mice, despite a robust increase in beta cell cross‐sectional area (as an index of beta cell mass). In contrast, high fat‐fed PKO mice had improved glucose tolerance compared to WT littermates, despite no increase in beta cell cross‐sectional area. These data suggest that either PKO mice are protected from diet‐induced insulin resistance or their beta cell function is enhanced to the extent to be able to compensate for systemic insulin resistance. Therefore, blocking the actions of Mig6 and thereby enhancing EGFR signaling could represent a novel strategy for preventing or delaying the progression to T2D.

Support or Funding Information

NIH grant DK099311 (PTF)
HMGB1 mediates sidestream cigarette smoke‐induced metabolic disruption
Authors: Taylor Oliver J. , Porter Margot E. , Reynolds Paul R. , Bikman Benjamin T.
Published: November 2023

High‐mobility group box 1 (HMGB1) is a protein released in response to various noxious stimuli, including cigarette smoke. Its pro‐inflammatory actions generally occur through activation of prominent pattern recognition receptors, namely RAGE and TLR4. We have previously found that both RAGE and TLR4 activation disrupt mitochondrial physiology and insulin signaling in skeletal muscle via ceramide accrual. Thus, the purpose of these studies was to determine whether HMGB1 is sufficient in mediating the metabolic disruption observed with sidestream smoke exposure and whether this effect is dependent on altered ceramide metabolism. Initially, we confirmed that HMGB1 was increased in skeletal muscle with chronic sidestream cigarette smoke exposure. To test the effect of HMGB1 directly, we found that HMGB1 treatment increased muscle cell ceramide levels and disrupted mitochondrial and insulin function, an effect that was alleviated with ceramide inhibition. Moreover, ceramide inhibition partially prevented reduced insulin and glucose tolerance in mice, and protected mitochondrial function in skeletal muscle. Altogether, these data suggest that HMGB1 may be a relevant signaling mediator in the metabolic response to cigarette smoke exposure.
DCD Donor Heart Pathophysiology and Preconditioning
Authors: Kearns Mark J , Cheung Anson , Bashir Jamil , Seidman Michael A , Boyd John H
Published: November 2023

Purpose

Organ donation after circulatory death (DCD) is increasingly proposed as a means of addressing organ supply/demand mismatch in cardiac transplantation. We previously demonstrated in a rat DCD protocol that there is no detectable myocardial necrosis in DCD hearts up to a warm ischemic time (WIT) of 16 minutes, but that left ventricular function is impaired. Furthermore, we identified several discriminatory biomarkers in DCD hearts in response to ischemia and reperfusion. The goal of the present study is to investigate the impact of increasing WIT doses, and the ability of a preconditioning agent (CpG, a toll‐like receptor 9 agonist) to mitigate injury in DCD hearts.

Methods

DCD hearts exposed to 5, 10, and 20 minutes of acirculatory arrest (with and without pre‐withdrawal CpG administration), beating heart controls, and positive ischemic controls were studied in rats. Left ventricular (LV) functional analyses were conducted in a normothermic, volume‐loaded mode using an ex‐vivo heart perfusion (EVHP) rig. Blinded histologic assessment was performed on trichrome‐stained LV cross‐sections. The relative expression of 4 mRNA transcripts (Myc, Jun, HMOX1, and HSP90aa1) was determined by QPCR in each group.

Results

DCD hearts demonstrated LV functional impairment, which increased with WIT dose. Short WITs weren't associated with irreversible injury, whereas longer WITs (e.g. 20min) were. An aggregate score using donor physiology correlated with the extent of LV dysfunction on EVHP. Furthermore, 4 mRNA biomarkers demonstrated expression profiles that correlated with structural injury and LV dysfunction across WIT groups. CpG oligonucleotides mitigated both functional and structural injury in DCD hearts compared with untreated groups.

Conclusions

DCD hearts exposed to short WITs incur functional impairment that is likely reversible, but the extent irreversibility increases with WIT dose. Donor withdrawal variables and mRNA biomarkers were helpful in predicting reversibility of cardiac injury. Critically, a simple pharmacologic agent (CpG) mitigated the degree of structural and functional injury in DCD hearts. This information will improve our ability to accurately determine clinical cutoffs for graft viability. Furthermore, our preconditioning approach raises the possibility of mitigating DCD‐related cardiac injury both prior to donor withdrawal and as a component of machine reperfusion ex‐vivo.

Support or Funding Information

Mark J. Kearns is supported by a CIHR Frederick Banting and Charles Best Doctoral Research Award, and a Heart & Stroke Robert Hayden Research Fellowship.
Acute Effects of Sugar‐Sweetened Beverage Consumption on Reactive Hyperemia in Young, Healthy Humans
Authors: Keller Joshua L , Kelsch Elizabeth A , Castellanos Diana Cuy , Crecelius Anne R
Published: November 2023

Recent studies have reported that chronic consumption of sugar‐sweetened beverages (SSBs) is associated with greater cardiovascular disease prevalence including hypertension, stroke and myocardial infarction. However, there are limited reports regarding the effects of acute consumption of SSBs on vascular function. Reactive hyperemia (RH) has been identified as a relevant test of microvascular function that is associated with cardiovascular disease risk. The purpose of this study was to test the hypothesis that consumption of an SSB would acutely impair microvascular responses, as tested by the RH protocol. Eight young healthy subjects were given 24 fluid ounces of placebo (water) or SSB (68g mixture of fructose and dextrose mimicking the composition of high fructose corn syrup, diluted in water). The order of drinks was counterbalanced between experimental visits. Prior to, immediately after, and 1 hour post‐beverage consumption, venous occlusion plethysmography was used to measure the forearm blood flow (FBF) during baseline and post‐ischemia (5 min) RH conditions. Whole blood [glucose] was determined via finger‐stick samples and rapid glucometry. Heart rate (HR; ECG) and blood pressure (BP; automatic sphygmomanometry) were monitored throughout the experiment. SSB consumption increased whole blood [glucose] from pre‐beverage (68±3 vs 104±6 mg/dl) whereas the placebo did not (69±3 vs 72±3 mg/dl). After 1 hour post‐beverage, [glucose] was near pre‐beverage values for both SSB (76±5 mg/dl) and placebo (75±4 mg/dl) conditions. A small decrease in peak RH FBF was observed 1 hour post‐SSB (27.6±2 vs 31.0±2.7 ml/dl/min; p=0.05); however, no other significant changes were observed. Interestingly, the dynamic response of the RH was altered such that the area of the curve (AUC) was slightly greater 1 hour post‐SSB vs immediately after beverage consumption (104.0±17 vs 84.5±17 ml). Increased systolic and diastolic BP was observed in the SSB condition following the drink and this persisted 1 hour post‐beverage. In general, HR was unchanged across all conditions. These findings indicate that young, healthy humans maintain microvascular functioning, as measured by RH following acute consumption of an SSB. Future studies should address at‐risk populations as well as whether chronic SSB consumption directly attenuates RH responses.
Effect of aging on the CO2 reactivity and blowout time in ocular vessels
Authors: Miyaji Akane , Ikemura Tsukasa , Hayashi Naoyuki
Published: November 2023

Ocular vessels are sensitive to changes in the partial pressure of arterial CO2 (PaCO2). It is well known that the resting blood flow in the ophthalmic artery decreases with age. However, no previous studies have investigated the existence of an age‐related change in the ocular CO2 reactivity; that is, the relative response of the blood flow to a given increase in PaCO2. It has been reported that a possible age‐related decline in the cerebral CO2 reactivity could be associated with vascular function including the blood supply and vessel stiffness, whereas the presence of an age‐related change in the cerebral CO2 reactivity is controversial. The objective of this study was to determine whether the CO2 reactivity in ocular and cerebral vessels changes with aging, and whether there is a relationship between the CO2 reactivity and vascular function in these vessels. We compared the CO2 reactivity responses in the middle cerebral artery (MCA) and optic nerve head (ONH) to a 3‐min inhalation of CO2‐rich air (5%) between 14 young (age 23±0 yr, mean±SD) and 11 middle‐aged (age 48±2 yr) healthy subjects. We examined the relationship in these vessels between the CO2 reactivity and blowout time (BOT), which is an index for assessing the ocular vascular function calculated from flow velocity waveform analysis and is reportedly correlated with aging. The CO2 reactivity in the ONH was significantly greater in the young group (1.0±0.1%/mmHg) than the middle‐aged group (0.6±0.1%/mmHg), but it did not differ in the MCA between the two groups (2.2±0.2%/mmHg and 2.7±0.4%/mmHg, respectively). In the ONH there was no correlation between the CO2 reactivity and BOT, while the CO2 reactivity and BOT were both significantly correlated with age (r=−0.45, p<0.05, and r=−0.54, p<0.05, respectively). In the MCA the BOT was not correlated with either age or CO2 reactivity. These results suggest that there are regional differences in the effect of aging on the CO2 reactivity, and that this reactivity is independent of the vascular function.

Support or Funding Information

JSPS KAKENHI grants: number 15J11556 to A.M. and number 26560396 to N.H.
Endothelin‐1 Receptor Responses during the Menstrual Cycle
Authors: Sebzda Kelly N , Wenner Megan M
Published: November 2023

Endothelin‐1 (ET‐1) is involved in regulating vascular function through two receptor subtypes: endothelin A (ET‐A) and endothelin B (ET‐B). Studies in animal and cell models suggest that ovarian hormones, such as estradiol (E2) and progesterone (P4), modulate differences in ET‐1 mediated vasomotor function. However, these mechanisms have not been comprehensively examined in humans. Therefore, the purpose of this study was to test the hypothesis that ovarian hormones would mediate microvascular vasodilation through an ET‐B receptor mechanism. We measured microvascular vasodilatory responses (% CVCmax) during local skin warming via laser Doppler flowmetry coupled with intradermal microdialysis perfusions of lactated Ringers (control), ET‐B (BQ‐788, 300nM), and ET‐A (BQ‐123, 500nM) receptor antagonists in ten young women (21±1 years, 23±1 kg/m2, 85±2 mmHg resting mean blood pressure) during the mid‐luteal (ML; days 20–25) and early follicular (EF; days 2–6) phases of the menstrual cycle. As expected, serum E2 (ML: 132.9±19.7 vs. EF: 51.1±10.6 pg/ml, P < 0.05) and P4 (ML: 6.8±1.5 vs. EF: 0.9±0.2 ng/ml, P < 0.05) levels were higher in women during the ML compared to the EF visit. ET‐B receptor blockade decreased vasodilatory responses during the ML phase (control: 91.0±1.9 vs. BQ‐788: 84.4±2.1% CVCmax, P < 0.05) but not during the EF phase (control: 88.5±2.4 vs. BQ‐788: 88.9±1.2% CVCmax, P = 0.88). ET‐A receptor blockade tended to decreased vasodilatory responses during the ML phase (control: 91.0±1.9 vs. BQ‐123: 87.7±1.8% CVCmax, P = 0.09) but not during the EF phase (control: 88.5±2.4 vs. BQ‐123: 92.2±2.4% CVCmax, P = 0.20). These data suggest that ET‐B receptors mediate vasodilation during the ML phase of the menstrual cycle, when both E2 and P4 are elevated. Additional studies are needed to determine the direct influence of E2 and P4 on ET‐B receptor function.

Support or Funding Information

Supported by NIH Grant U54 GM 104941 01A1 and University of Delaware Research Foundation
Low‐dose Aspirin During Gestation Promotes Vascular Dysfunction and Does Not Ameliorate Maternal Hypertension in Rats Exposed to Innate Immune System Activation
Authors: Jaini Paresh Atu , Nguyen An , Valdes Melissa , Osikoya Oluwatobiloba , Goulopoulou Styliani
Published: November 2023

Background

Daily low‐dose aspirin after 12 weeks of gestation is recommended as a preventive intervention for women at high risk for preeclampsia, a hypertensive disorder of pregnancy with high rates of maternal and fetal mortality and morbidity. Activation of the innate immune system during pregnancy is implicated in the development of preeclampsia. Maternal exposure to synthetic CpG oligonucleotides (CpG ODN, specific ligand of the innate immune receptor Toll‐like receptor 9) induces maternal hypertension, vascular dysfunction, and upregulation of cyclooxygenase enzymes in pregnant rats.

Hypothesis

We hypothesized that maternal treatment with low‐dose aspirin during gestation would ameliorate TLR9‐induced hypertension and vascular dysfunction in pregnant rats.

Methods

Pregnant Sprague‐Dawley rats were treated with a synthetic CpG ODN (ODN2395) or vehicle on gestational day (GD) 14, 16, and 18. Aspirin treatment (or control) started on GD10 and continued throughout gestation for all groups [control (no treatment), ODN2395 (300 μg), aspirin (1.5 mg/kgBW), aspirin+ODN2395]. Blood pressure was measured on GD19 using the tail cuff method and mesenteric resistance artery (MES) function was assessed on GD21 using wire myography.

Results

ODN2395‐treated rats had higher blood pressure on GD19 compared to vehicle‐treated dams and aspirin did not ameliorate ODN2395‐induced hypertension (control: 97 ± 0.4 mmHg, ODN2395: 121 ± 7 mmHg, aspirin: 101 ± 5 mmHg, aspirin+ODN2395: 121 ± 7 mmHg, p<0.05). Aspirin treatment increased MES sensitivity to PE (pEC50, ODN2395: 5.6 ± 0.1 vs. aspirin+ODN2395: 5.9 ± 0.1, p<0.05) and reduced sensitivity to acetylcholine (ACh) in ODN2395‐treated rats (pEC50, ODN2395: 7.6 ± 0.1 vs. aspirin+ODN2395: 7.0 ± 0.1, p<0.05). Fetal and placental weights did not change in response to any treatment.

Conclusion

Treatment with low‐dose aspirin throughout gestation did not prevent the development of TLR9‐induced maternal hypertension, augmented vascular sensitivity to α1‐adrenergic receptor activation, and attenuated endothelium‐dependent dilation in rats exposed to innate immune system activation. The use of aspirin during gestation should be considered with caution in clinical cases associated with innate immune system‐induced pregnancy complications.

Support or Funding Information

American Heart Association, University of North Texas Health Science Center, Texas College of Osteopathic Medicine Summer Research Fellowship