bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of Sex Differences in Vascular Density in a Rodent Model of Traumatic Brain Injury Using Steady‐State Contrast Enhanced Magnetic Resonance Imaging
Authors: Mayo Jamie Nadine , Wright David , Phillips Roree , O'Brien Terence J. , Shultz Sandy
Published: November 2023

Objective

The mechanisms by which female rats fare better then male rats post‐TBI remain unclear. Changes in vascular density and other measures of new blood vessel growth, angiogenesis, play a role in recovery post‐TBI. Vascular function has been hypothesized to be involved in sex differences in recovery post‐TBI. It is unknown if there are sex differences in injury induced changes in vessel density post‐TBI. The hypothesis was tested that females would have greater vessel density 48 hours post‐TBI than males, and that they would outperform males on motor coordination tasks one week after injury.

Methods

Adult male and female rats were subjected to a severe TBI using a fluid percussion injury model. Steady‐state contrast enhanced MRI (SSC‐MRI, 15 mg/kg of ferumoxytol) was used to assess injured cortex vessel density in vivo 48 hours post‐injury. Motor coordination was assessed one week post‐injury using the balance beam.

Results

Injury and sex were a significant source of variation (p < 0.05) in regards to motor coordination performance. Injured male rats had a 2.4‐fold increase in beam traversal time (p < 0.05) as compared to male shams. No statistical difference was found between injured and sham females (p > 0.05). In regards to the SSC‐MRI results, 48 hours post‐ injury both sex and injury were a significant source of variation in vessel density within the ipsilateral cortex (p < 0.05). Injured males had a 0.84 fold decrease in vessel density as compared to male shams (p < 0.05). Injured females did not have a significant difference in vessel density as compared to female shams (p > 0.05).

Conclusions

The greater vessel density in injured females as compared to injured males may contribute to sex differences in motor coordination post‐TBI. SSC‐MRI could be used in future clinical studies in order to elucidate the temporal changes in cortical vessel density post‐TBI. A better understanding of sex differences in vascular responses post‐injury could aide in ensuring that the development of therapies targeting angiogenesis post‐TBI will be appropriate for both male and female patients.
Recovery of Vasomotor Control in Microvascular Resistance Networks of Regenerating Skeletal Muscle
Authors: Fernando Charmain A , Cornelison D W , Segal Steven S
Published: November 2023

The microcirculation is disrupted during degeneration of injured skeletal muscle (skm). While regeneration of skm fibers is well studied, little is known about recovery of the microcirculation during skm regeneration. Our working hypothesis is that restoration of microvascular structure and function is integral to skm regeneration. In anesthetized male C57BL/6J mice (4 months, n=4/group), the gluteus maximus muscle (GM) was injured by local injection of barium chloride solution (1.2%), causing degeneration/regeneration of affected muscle fibers. At 5 days (d) post‐injury (PI), microvascular perfusion was restored as determined by intravital imaging of fluorescent plasma (FITC‐dextran, 70 kDa) in the regenerating GM. Functional integrity of the resistance network was evaluated at 5, 10 and 21d PI vs control by measuring internal diameter (ID) of feed arteries (FA), first‐ (1A), second‐ (2A) and third‐order (3A) arterioles supplying the GM. Maximal IDs (topical sodium nitroprusside, 10−4 M) decreased as branch order increased (P<0.05) but were not different across time points within each branch order. Resting IDs of all branch orders were greater (P<0.05) than control at 5d and 10d PI, particularly in FA and 1A, then recovered to control by 21d, as did spontaneous tone. At each time point, responses to increasing agonist concentration were evaluated from 10−9 to 10−5 M. Vasoconstriction to α1‐adrenoreceptor (AR) stimulation (phenylephrine) was attenuated at 5d across branch orders and recovered to control by 21d. Vasoconstriction to α2AR stimulation (UK14304) was ~50% less (P<0.05) than to α1AR stimulation and was not different across respective time points vs control. Vasodilation to acetylcholine was negligible at 5d and increased progressively at 10d and 21d PI (P<0.05) for all branch orders, with greatest efficacy in 3A. Thus myogenic tone and smooth muscle reactivity to α1AR stimulation recover in parallel while endothelium‐dependent vasodilation recovers progressively through 21d PI. Resolving the time course for restoration of vasomotor control in regenerating skm provides novel perspective towards developing therapeutic strategies for restoring skm function during recovery from injury.

Support or Funding Information

Mulligan Professorship, NIH RO1AR067450, NIH R37HL041026
Exercise Recovers Loss of Microvessel Density due to Lowered Nitric Oxide Bioavailability
Authors: Branyan Kayla , Ashman Melissa , Pitzer Christopher , Chantler Paul , Olfert Mark
Published: November 2023

Microvessel rarefaction is an indicator of a challenged or disease state in both humans and animal models. The loss of peripheral microvessel density is associated with the metabolic syndrome (MetS). Decreased microvessel density significantly increases the risk for vascular dysfunction and peripheral vascular disease (PVD). Nitric oxide (NO) has been shown to have pro‐angiogenic properties, which suggests that improvement of NO bioavailability in MetS can augment microvessel rarefaction. Exercise, when used as an intervention during MetS, can improve NO bioavailability. The initiating factors for microvessel rarefaction still remain unclear and whether or not exercise can reverse the rarefaction associated with MetS needs to be determined. To examine the correlation between loss in microvessel density and decrease in nitric oxide (NO) bioavailability, capillary to fiber ratio was measured in the soleus and gracilis muscles of obese Zucker rats (OZRs), a model of MetS, and in lean healthy Zucker rats (LZRs). NO levels were measured in stimulated aortas of both groups. OZRs and LZRs were separated into sedentary and exercise training groups (consisting of treadmill running 1 hr/d, 5 d/wk for 8 weeks at 70% of their max running speed). The gracilis arteriole was isolated and then cannulated in a pressurized vessel myobath and exposed to increasing doses of acetylcholine (ACh) to test endothelium‐dependent dilation. Capillary density in the soleus was significantly lower by 15% in OZRs compared to LZRs (p<0.05), and exercise training did not have an effect. In the gracilis, exercise improved capillary density in both OZRs and LZRs. The difference in capillary density between OZR and LZR controls in the gracilis suggest a genotypic disparity. NO bioavailability was significantly decreased in sedentary OZR compared to sedentary LZR, and exercise augmented NO in both groups (p<0.05). Gracilis arteriole dilation response was lower in OZR than LZR (p<0.01) but exercise improved dilation in OZR to almost LZR control values. These results suggest that there is a correlation between decreased NO bioavailability and the loss of peripheral microvessel density, and that exercise may at least partially, depending on the muscle type, reverse this effect and recover arteriole reactivity.

Support or Funding Information

The authors gratefully acknowledge the support by National Institute of Health (5P20GM109098)
IGF‐1 Receptor on Leukocytes Potentiates Western Diet‐induced Inflammatory Response and Progression of Atherosclerosis in ApoE‐Deficient Mice
Authors: Wang Meifang , Higashi Yusuke , Wang Derek , Yu Hong , Delafontaine Patrice , Korthuis Ronald
Published: November 2023

Atherosclerosis is the principal underlying cause of most cardiovascular disease‐related deaths, the leading cause of mortality in the US. Insulin‐like growth factor‐1 (IGF‐1), the primary mediator of the effect of growth hormone on developmental growth, is expressed in vascular cells and monocytes/macrophages. However, IGF‐1's role in atherosclerosis is unknown. Our previous studies showed that lowered circulating IGF‐1 increases atherosclerosis progression in atherosclerosis‐prone apolipoprotein E‐deficient mice (ApoE−/−) while increased circulating IGF‐1 downregulates expression of vascular pro‐inflammatory cytokines, reduces systemic and vascular oxidant stress, and decreases atherosclerosis progression. We also demonstrated that that the aforementioned inflammatory phenotype is potentiated in mice with double knockout of ApoE and IGF1 receptor (IGF1R), where IGF1R expression was selectively deleted in monocyte/macrophages (ApoE−/− LC), and that consumption of a Western diet enhanced monocyte‐endothelial interactions in these double knockout mice compared to wild type IGF‐1R/ApoE−/−‐flox mice (ApoE−/−‐FIR). Here, we examined similar endpoints in an ApoE/IGFR1−/− model in which IGF1R‐expression was selectively deleted in endothelial cells (ApoE−/− VF), and again examined the possible interaction of the effect of these knockouts with consumption of a Western diet, comparing ApoE−/− VF with IGF‐1R ApoE−/−‐flox mice (ApoE−/−‐FIR). We compared baseline Leukocyte Rolling (LR) and Leukocyte Adhesion (LA) in both these mice fed either normal mouse chow (NC) or Western diet (WD). There was significant increase in the pro‐inflammatory phenotype in both NC fed mice (ApoE−/− VF NC and ApoE−/−‐FIR NC) compared to C57BL6 wild type mice. Ingestion of a Western Diet markedly enhanced LR and LA on both ApoE−/− VF animals (ApoE−/− VF WD) and ApoE−/− ‐FIR animals (ApoE−/− ‐FIR WD), however. This finding indicates that the pro‐inflammatory phenotype enhanced by WD in ApoE−/− VF and FIR mice is not further enhanced by specific IGF1R deficiency in endothelial cells. We conclude that IGF‐1 receptor on leukocytes, but not endothelial cells, potentiates the inflammatory phenotype induced by Western Diet in ApoE‐deficient mice.

Support or Funding Information

Supported by NIH: R01‐HL59976, RO1 HL070241 and HL 080682, R01‐AA22108.
Interleukin‐12 Disruption Protects Myocardium from Ischemia‐Reperfusion Injury in Type 2 Diabetic Mice
Authors: Mali Vishal , Tennant Will , Haddox Samuel , Matrougui Khalid , Belmadani Souad
Published: November 2023

Introduction

Myocardial infarction (MI) is the most common cause of mortality in diabetes. In the present study, we determined the in vivo role and mechanism for interleukin‐12 (IL‐12) in myocardial infarction in type 2 diabetic mice.

Methods and Results

C57/Bl6, IL‐12−/−, db−/db−, and db−/db−IL‐12−/− were subjected to myocardial ischemia for 40 minutes and followed by reperfusion (I/R) injury for 24 hours. I/R injury induces a significant myocardial infarct in C57/Bl6 but substantially greater in db−/db− mice. Interestingly, IL‐12−/− and db−/db−IL‐12−/− are protected against I/R injury as indicated by the small infarct size. The IL‐12 disruption protective effect involves endoplasmic reticulum (ER) stress, inflammation, AMPKinase and Akt signaling. Thus, phosphorylated AMPKinase and Akt were enhanced in the heart from IL‐12−/− and db−/db−IL‐12−/− mice subjected to I/R injury compared to C57/Bl6 and db−/db− subjected to I/R injury. Moreover, after I/R injury, inflammation, assessed by the infiltration of macrophages and leukocytes into the heart, and ER stress CHOP were reduced in IL‐12−/− and db−/db−IL‐12−/− compared to C57/Bl6 and db−/db− mice.

Conclusion

The present study highlighted the in vivo essential role for IL‐12 disruption in the cardio‐protection against I/R injury in type 2 diabetes. The protective mechanism of IL‐12 disruption involves ER stress, inflammation, AMPKinase, and Akt pathways.
Cathepsin B Mediated Degradation of Jejunal Epithelial Tight Junction Proteins during the Early Stage of Hemorrhagic Shock
Authors: Klein Nikolas Philipp , Courelli Asimina , Schmid‐Schoenbein Geert
Published: November 2023

Hemorrhagic shock has been strongly linked to intestinal permeability — a hallmark of intestinal epithelial barrier breakdown — whose increase is associated with reduced function of intercellular adhesion proteins. The early effects of hemorrhagic shock (HS) on junctional epithelial proteins and the potential mechanisms of increased intestinal permeability have not been fully investigated. We investigate three junctional proteins responsible for epithelial cell adhesion in the jejunum: Claudin‐3, Occludin, and E‐Cadherin. We also investigate the role of Cathepsin B, an epithelial lysosomal cysteine protease, in the degradation of those adhesion protein. We hypothesize that Cathepsin B cleaves the extracellular domain of the adhesion molecules in HS. 16 Sprague‐Dawley rats were divided into 4 groups of 4 animals: Control, HS (MAP = 35mmHg for 30 min without blood volume reconstitution), HS with enteral pre‐treatment by Golytely as a fluid carrier, and HS with enteral pre‐treatment by Golytely and CA074 (Cathepsin B inhibitor at 15mg/kg). Jejunum was collected for Western Blot and immunohistochemistry of Claudin‐3, Occludin, and E‐Cadherin. Both Western Blot and immunohistochemistry results showed a decrease in levels of Claudin‐3 (HS p‐value: 0.052; HS with Golytely p‐value: 0.026), Occludin (HS p‐value: 0.011; HS with Golytely p‐value: 0.0036), and E‐Cadherin (HS p‐value: 0.013; HS with Golytely p‐value: 0.00137) in HS and HS pretreated with Golytely groups when compared to Control. Addition of Cathepsin B inhibitor resulted in maintenance of junctional proteins in the jejunum after HS. These results indicate that active Cathepsin B is released early during HS and causes a reduction of cell‐cell adhesion proteins Claudin‐3, Occludin, and E‐Cadherin, leading to increased intestinal permeability.

Support or Funding Information

NIH Grant: GM 85072
Chloropalmitate increases solute permeability and VE‐cadherin internalization in human microvascular endothelial cells
Authors: Kalogeris Theodore , Birkhead Travis Michael , Ford David , Korthuis Ronald
Published: November 2023

Chlorinating species generated through the activity of neutrophil/macrophage myeloperoxidase have been found to produce various types of chlorinated lipids under inflammatory conditions, and these lipids In turn have been implicated as potential contributing factors in the inflammatory phenotype seen after ischemia/reperfusion. In previous studies in cultured microvascular endothelial cells, we documented increases in cortical stiffness and cytoskeletal reorganization in cells exposed to chloro‐palmitate. In the present studies we examined whether chlorolipids could also affect endothelial barrier function. Human microvascular endothelial cells (HMEC‐1) grown to confluence on 0.4 μm transwell inserts were incubated with 2‐chlorohexadecanal (chloro‐fatty palmitaldehyde, Cl‐HDA) or 2‐chlorohexadecanic acid (chloro‐palmitate, CL‐FA), or their non‐chlorinated lipid precursors as controls, and the permeability of the monolayer to FITC‐albumin was calculated from measured albumin flux and transwell albumin concentration gradient using Fick's law. Compared with control treatments (palmitate, ethanol [vehicle]), Cl‐FA produced a 3–4‐fold increase in permeability which persisted over at least 4 h, similar to the permeability effect of the positive control agent, TNFα, while Cl‐HDA's effect was not significantly different from control. In followup studies, we examined the effect of the aforementioned treatments on VE‐cadherin distribution using a trypsin‐protection assay. Treatment with Cl‐FA or Cl‐HDA protected cell surface VE‐cadherin from trypsin‐induced degradation, consistent with chlorolipid‐induced VE‐cadherin internalization. These data support the hypothesis that Cl‐FA induces endothelial barrier dysfunction associated with increased cell stiffness, and reorganization of the cortical cytoskeleton and adherens junctions. Our results also suggest a dissociation between adherens junction integrity and permeability in the case of Cl‐HDA.

Support or Funding Information

N.I.H. GM‐115553, AA‐22108
Cardioprotective effects of hydroalcoholic extract of Terminalia capatta in isolated perfused rat hearts.
Authors: Torres‐Tirado Jose David , Pérez Gabriela , Sanchez Eugenia , Estrada Alejandro , Zacarias Alejandra , Leon Angel
Published: November 2023

The use of traditional medicinal plants in prevention and treatment of different illnesses is a comun practice in the world. Mexico has a long traditional plants applications since ancient times. Terminalia catappa is used in the empirical treatment of skin illness, digestive diseases and in a recent study the seed extract show cardio protective effects. The aim of this work was to test the cardioprotective effect of hydroalcoholic extract of Terminalia capatta leaves. The extract was oral administrated one time a day in wistar rats during seven days after its were sacrificed to test ten min ischemia and ten min reperfusion in Langendorff isolated heart technique. Results showed an important decrease in loss of contraction of the left ventricular pressure of 20 % vs control. The histology showed an 16% decrease in the infarct size vs control. Terminalia capatta extract showed an important cardioprotective effects in our model.

Support or Funding Information

This work was supported by grants from UASLP (FAI 2014 to JTT and 2016 to GP)
Calreticulin localized to the myoendothelial junction mediates heterocellular communication in resistance arteries
Authors: Biwer Lauren A , Good Miranda E , Hong Kwangseok , Patel Rahul , Agrawal Neha , Looft‐Wilson Robin , Sonkusare Swapnil , Isakson Brant E
Published: November 2023

Regulated heterocellular communication depends on bidirectional signaling that occurs at myoendothelial junctions (MEJs), which are composed of endothelial cell (EC) extensions that make direct cytoplasmic contact with smooth muscle cells (SMC). The MEJ is the anatomical hallmark of resistance arteries and is a key signaling microdomain with endoplasmic reticulum (ER) as we demonstrate via expression of the ER retention signal –KDEL, and ER tracker in a live artery, both of which localize to holes in the internal elastic lamina. Presumably, the expression of ER in the MEJ could facilitate localized Ca2+ events at MEJs generated in response to second messengers from EC agonists or smooth muscle (SMC) agonists (by movement of IP3 or Ca2+ through gap junctions to the EC extensions composing MEJs). To determine what proteins may regulate the spatially localized Ca2+ events at the MEJ, we used an iTRAQ proteomic screen on in vitro MEJs and found significant enrichment of calreticulin. Calreticulin is a calcium binding protein that has been shown to bind ~50% of ER calcium. We confirmed the presence of calreticulin at MEJs using third‐order mesenteric arterioles. Thus, we hypothesized that deletion of EC calreticulin could alter Ca2+ events at the MEJ that were generated after application of EC or SMC agonists. For this reason we created an EC specific calreticulin conditional knockout mouse (Calreticulinfl/fl/CDH5‐CreERT2+). Initial experiments were performed in mice between 10–20 weeks old. In these experiments, carbachol (CCH; EC specific) or phenylephrine (PE; SMC specific) was added to third order mesenteric arteries and the Ca2+ events were quantified. The control mice had a robust increase in Ca2+ events at MEJs after PE stimulation, but Calreticulinfl/fl/CDH5‐CreERT2+ mice had a significantly reduced number of Ca2+ events. Furthermore, Calreticulinfl/fl/CDH5‐CreERT2+ mice had significantly enhanced constriction to PE, demonstrating the importance of calreticulin at the MEJ in the PE response. Conversely, CCH stimulation showed no differences in Ca2+ events and no difference in dilation to CCH in the Calreticulinfl/fl/CDH5‐CreERT2+ mouse. This polarized response in Ca2+ events at MEJs in Calreticulinfl/fl/CDH5‐CreERT2+ endothelium was lost in mice aged >75 weeks. These results demonstrate: 1) the enrichment of calreticulin to MEJs, 2) polarized activation of EC Ca2+ events is based upon whether the agonist is EC (apical) or SMC (MEJ) in origin and 3) aging alters how calreticulin polarizes Ca2+ events after agonist stimulation. Overall, we believe EC calreticulin can regulate Ca2+ events generated in responses to heterocellular communication.

Support or Funding Information

National Institutes of Health HL088554, HL120840, T32 HL007284, and American Heart

Association 14PRE20420024
Administration of Prostacyclin Mediates Cutaneous Vasodilation but not Sweating in Young and Older Females: Roles for Nitric oxide and KCa channels
Authors: Fujii Naoto , McNeely Brendan D , Kenny Glen P
Published: November 2023

Our recent report indicated that although prostacyclin does not mediate sweating in young and older males, it does modulate cutaneous vasodilation in both groups to a similar extent (Fujii et al. J Physiol 594 (21), 6419–6429, 2016). The same study also suggested that nitric oxide synthase (NOS) and calcium‐activated potassium (KCa) channels contribute to the prostacyclin‐induced cutaneous vasodilation in young males but, these contributions are diminished in older males. In our current study, we sought to evaluate whether the same responses are observed in young and older females. In young (25±4 years) and older (60±6 years) females (10 per group), cutaneous vascular conductance (CVC) and sweat rate were evaluated at four intradermal forearm skin sites: 1) Control, 2) 10 mM L‐NNA, a non‐specific NOS inhibitor, 3) 50 mM tetraethylammonium (TEA), a non‐specific KCa channel blocker, and 4) 10 mM L‐NNA + 50 mM TEA. All fours sites were co‐administered with prostacyclin in an incremental manner (0.04, 0.4, 4, 40, 400 μM each for 25 min). Our results demonstrated that both NOS and/or KCa channels contribute to prostacyclin‐induced cutaneous vasodilation for young and older females. This is based on our observations that the increased CVC due to prostacyclin administration was attenuated by TEA (0.4 μM) and a combination of L‐NNA and TEA (at all concentrations tested) in young females. Additionally, in older females the increased CVC due to prostacyclin administration was also reduced by separate administration of either L‐NNA or TEA (0.04–4 μM), and a combination of both agents (0.04–40 μM) (all P≤0.05). Surprisingly, increases in CVC in response to 0.04–4 μM prostacyclin were greater in older relative to young females (all P≤0.05), and these age‐related differences were diminished when both L‐NNA and TEA were simultaneously administered (all P>0.05). No effect on sweat rate was observed in either group (all concentrations, P>0.05). We conclude that while prostacyclin does not mediate sweating, it modulates cutaneous vasodilation to a greater extent in older relative to young females. Wherein, this modulation is likely due to NOS‐ and KCa channel‐dependent mechanisms.

Support or Funding Information

This study was supported by the Natural Sciences and Engineering Research Council of Canada (Discovery grant, RGPIN‐06313‐2014; Discovery Grants Program ‐ Accelerator Supplement, RGPAS‐462252‐2014). Funds held by Dr. Glen P. Kenny.
The Extracellular Matrix Proteoglycan Decorin is Upregulated by Endothelial Cells During Inflammation and Contributes to Blood‐Brain Barrier Dysfunction
Authors: Hoettels Brian A. , Wertz Travis S. , Birk David E. , Oxford Julie T. , Beard Richard S.
Published: November 2023

The blood‐brain barrier (BBB) selectively regulates solute flux and protects the brain from bloodborne neurotoxic substances. Endothelial cells (ECs) forming the BBB establish highly restrictive cell‐cell contacts known as tight junctions (TJs), which aid in sealing paracellular clefts. The TJ protein claudin‐5 (CLDN5) is the primary constitutive protein responsible for the restriction of paracellular permeability to small molecules and the level of CLDN5 expression has been positively correlated with endothelial barrier tightness. Situated below the ECs is the extracellular matrix (ECM) which not only helps anchor the ECs, but also participates in ECM‐EC regulatory signaling. Under normal conditions, certain ECM proteins, such as collagens, have been shown to enhance the barrier properties of the BBB, whereas during inflammation other ECM proteins can contribute to BBB dysfunction, although the particulars of these mechanisms are still yet to be fully understood. In certain neuroinflammatory diseases, such as multiple sclerosis (MS), the upregulation of the ECM small leucine‐rich repeat proteoglycan (SLRP) decorin has been identified within the perivascular space of vessels in the inflammatory lesions of MS patients. The cellular origin of this decorin release and its potential impact on the BBB, however, has yet to be determined. In this study, we used murine models of inflammation in vivo and in vitro to determine a spatial‐temporal relationship between BBB dysfunction, CLDN5 loss, and increased decorin accumulation in the ECM. Consistent with the MS lesions, we identified increased decorin expression in mouse cerebellar microvessels post‐experimental autoimmune encephalomyelitis (EAE) induction as well as in primary brain microvascular endothelial cells (BMVECs) treated in vitro with inflammatory cytokines. Additionally, we evaluated the effect of different ECM‐EC interactions on CLDN5 expression and BMVEC monolayer integrity in vitro. Primary BMVECs cultivated on collagen‐coated surfaces showed a decrease in baseline solute permeability and increased CLDN5 expression compared to those on non‐coated surfaces, with those on collagen type IV‐coated surfaces showing a greater decrease in baseline permeability and CLDN5 expression compared to those on type I‐coated surfaces. However, surfaces containing decorin resulted in increased monolayer permeability and decreased CLDN5 expression, while matrices containing both decorin and type IV collagen resulted in abrogation of the observed enhancement of monolayer permeability and CLDN5 expression by type IV collagen. Taken together, our data suggest that ECs themselves may be the source of decorin accumulation in perivascular inflammatory lesions and that inflammatory‐induced decorin upregulation may perpetuate BBB dysfunction.

Support or Funding Information

Supported by NIH P20 GM109095, 2P20GM103408.
Alterations to Venular Function in Skeletal Muscle with Metabolic Syndrome
Authors: Lemaster Kent , Farid Zahra , Jackson Dwayne , Goldman Daniel , Brock Robert , Frisbee Jefferson
Published: November 2023

While the overwhelming majority of research into vascular outcomes of the metabolic syndrome have focused on the arterial/arteriolar and capillary levels, investigation into venular function and how development of metabolic syndrome impacts responses has received little attention. Using the in situ cremaster muscle of obese Zucker rats (OZR) as compared to control leans (LZR), we determined indices of venular network structure as well as vessel structure and function. At ~17 weeks of age, skeletal muscle post‐capillary venular density was reduced by ~20% in LZR vs. OZR, although there was no evidence of remodeling of the wall of individual venules impacting passive diameter. Initial basal diameter and blood flow within individual venules of ~25 mm and ~ 60 mm diameter was similar between LZR and OZR, although an increased variability was evident. Dilator responses to acetylcholine were consistently blunted in venules of OZR vs. LZR, and this reflected multiple effects: including an oxidant stress‐based loss of venular nitric oxide bioavailability, an increased constrictor tone from TxA2 and an increased constrictor tone mediated via the α1 adrenergic receptor. Impaired dilator responses were also coupled to vessels where increased leukocyte adhesion/rolling was clearly present. Venular constrictor responses between LZR and OZR were comparable for angiotensin II, endothelin and α2 adrenoreceptor stimulation, although responses to α1 adrenoreceptor stimulation were mildly elevated, but with increased variability. In response to field stimulation of the cremaster muscle (0.5 or 1/s, 400 ms duration, 60 Hz within train, 7V), venular dilator and hyperemic responses to contraction were blunted in OZR vs. LZR, but responses at the higher frequency were similar in magnitude. These results suggest that alterations in venular function may contribute to dysregulation of flow distribution in skeletal muscle with metabolic syndrome and impair parameters of mass transport and exchange.

Support or Funding Information

National Institutes of Health; American Heart Association
Adipose Tissue VEGF‐D Overexpression Increases Lymphatic Vessel Density and Protects Against Inflammation and Insulin Resistance in Obesity
Authors: Lammoglia Gabriela M , Barajas Sheridan E , Reyna Andrea J , Rutkowski Joseph M
Published: November 2023

Lymphatic vessels modulate tissue fluid balance and inflammation and provide a conduit for endocrine and lipid transport: all mechanisms that could potentially regulate adipose tissue physiology in obesity. The growth of new lymphatic vessels in is mediated through vascular endothelial growth factor receptor‐3 (VEGFR‐3) signaling. We took advantage of the unique binding of murine VEGF‐D to VEGFR‐3 and generated mice capable of inducible, tissue‐specific expression of murine VEGF‐D under a tightly‐controlled tetracycline response element (TRE) promoter to stimulate lymphangiogenesis. Crossed with adipocyte‐specific adiponectin‐rtTA mice (Adipo‐VD), VEGF‐D overexpression by adipocytes induced de novo lymphangiogenesis in murine white adipose tissue and a massive expansion of brown adipose tissue lymphatics. Upon removal of the doxycycline stimulus, VEGF‐D expression returned to normal and the expanded adipose tissue lymphatics regressed. The new white adipose lymphatic network provided a route for increased glycerol flux during lipolysis. As a chemokine, VEGF‐D expression recruited macrophages and caused fibrosis in white adipose tissue on chow diet, but no negative systemic metabolic consequences were identified. On high fat diet feeding, Adipo‐VD gained equivalent weight and adiposity as littermate controls, but were markedly more insulin sensitive. White adipose tissues in Adipo‐VD mice were full of lymphatics, but exhibited less fibrosis, contained fewer crown‐like structure macrophages, and displayed increased UCP‐1 RNA and protein. Infusion of CD45.1+ leukocytes into CD45.2+ Adipo‐VD mice identified fewer immune cells accumulating in the Adipo‐VD subcutaneous adipose with more trafficking out and into the inguinal lymph node. The increased lymphatic density thus reduces local inflammation in adipose tissue, manifesting as decreased liver triglyceride, increased insulin sensitivity, and overall healthier obesity highlighting the roles of lymphatic in regulating inflammatory pathologies and VEGFR‐3 signaling as a potential target in the metabolic syndrome.
Polyethylene Glycol Camouflaged Earthworm Hemoglobin
Authors: Cabrales Pedro , Jani Vivek
Published: November 2023

Nearly 21 million components of blood and whole blood and transfused annually in the United States, while on average only 13.6 million units of blood are donated. As the demand for Red Blood Cells (RBCs) continues to increase due to the aging population, this deficit will be more significant. Despite decades of research to develop hemoglobin (Hb) based oxygen (O2) carriers (HBOCs) as RBC substitutes, there are no products approved for clinical use. Lumbricus terrestris erythrocruorin (LtEc) is the large acellular O2 carrying protein complex of the earthworm Lumbricus terrestris. LtEc is an extremely stable protein complex, resistant to autoxidation, and capable of transporting O2 to tissue when transfused into mammals. These characteristics render LtEc a promising candidate for the development of the next generation HBOCs. LtEc has a short half‐life in circulation, limiting its application as a bridge over days, until blood became available. Conjugation with polyethylene glycol (PEG‐LtEc) can extend LtEc circulation time. This study explores PEG‐LtEc pharmacokinetics and pharmacodynamics. To study PEG‐LtEc pharmacokinetics, hamsters instrumented with the dorsal window chamber were subjected to a 40% exchange transfusion with 10 g/dL PEG‐LtEc or LtEc and followed for 48 hours. To study the vascular response of PEG‐LtEc, hamsters instrumented with the dorsal window chamber received multiple infusions of 10 g/dL PEG‐LtEc or LtEc solution to increase plasma LtEc concentration to 0.5, then 1.0, and 1.5 g/dL, while monitoring the animals' systemic and microcirculatory parameters. Results confirm that PEGylation of LtEc increases its circulation time, extending the half‐life to 70 hours, 4 times longer than that of unPEGylated LtEc. However, PEGylation increased the rate of LtEc oxidation in vivo. Vascular analysis verified that PEG‐LtEc showed the absence of microvascular vasoconstriction or systemic hypertension. The molecular size of PEG‐LtEc did not change the colloid osmotic pressure or blood volume expansion capacity compared to LtEc, due to LtEc's already large molecular size. Taken together, these results further encourage the development of PEG‐LtEc as an O2 carrying therapeutic.

Support or Funding Information

Funding: This work was supported by NIH grants from the Heart Lung and Blood Institute, P01‐HL110900, R56‐HL123015, and R01‐HL126945. Authors declare no competing financial interests.
Influence of Ischaemia‐Reperfusion Injury on the Subsequent Response to Local Heating in Non‐Glabrous and Glabrous Skin of the Index Finger
Authors: McGarr Gregory W. , Hodges Gary J. , Cheung Stephen S.
Published: November 2023

Ischaemia‐reperfusion (I‐R) injury has been implicated in the pathophysiology of many clinical conditions including frostbite and non‐freezing cold injuries. The increased oxidative stress and inflammation associated with I‐R injury may contribute to impaired restoration of blood flow and tissue recovery in the hands and feet during rewarming through alterations in neurovascular and/or endothelial function. This may be reflected in an altered blood flow response to local skin heating, which is characterized by an initial vasodilatory peak that is primarily controlled by an axon reflex, followed by a secondary plateau that is influenced mainly by nitric oxide release from the endothelium. In ten participants we examined the responses to local heating in non‐glabrous and glabrous skin of the index finger following 20 minutes of ischaemia induced by inflating (220 mmHg) a standard blood pressure cuff around the arm and compared these with a time‐matched sham (non‐ischaemia) trial. Red blood cell flux, an index of skin blood flow, was examined using laser‐Doppler probes with integrated local heating units. Flux values were then divided by mean arterial pressure and reported as cutaneous vascular conductance (CVC). Local heating was initiated prior to ischaemia and 20 minutes after the release of cuff occlusion at separate skin sites. At each site local temperature was controlled at 33°C and then increased (3°C min−1) to 42°C and held for ~30 min. Following this, local temperature was increased to 44°C for 20 min. No changes were found in CVC for the initial peak and plateau phases or when heating to 44°C following ischaemia in either skin type (all P>0.05). However, the kinetics of the response were significantly altered by I‐R. Vasodilatory onset was delayed by 24% and 19% for non‐glabrous and glabrous skin, respectively (both P<0.05). Time to initial peak was also delayed by 17% and 11% in non‐glabrous and glabrous skin, respectively, although only the delay in non‐glabrous skin was significant (P<0.05). We conclude that 20 minutes of I‐R injury significantly impairs the kinetics of the response to a rapid, non‐painful local heating stimulus in both non‐glabrous and glabrous skin of the index finger, suggesting an alteration in neurovascular control. This delayed response may be associated with a higher temperature threshold for initiating vasodilation post‐ischaemia and could lead to persistent hypoxic stress during rapid rewarming in association with an increase in local skin temperature, coupled with insufficient tissue perfusion.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada: Discovery Grant (227912‐12, S.S. Cheung)
Human Skeletal Muscle Feed Artery Function: Role of the Advanced Glycation End‐Products Receptor (RAGE)
Authors: Park Soung Hun , Kwon Oh Sung , Andtbacka Robert H. I. , Hyngstrom John R. , Richardson Russell S.
Published: November 2023

Background

Aging is associated with impaired endothelium‐dependent dilation in human skeletal muscle feed arteries (SMFAs), vessels recognized to have regulatory function. Although growing evidence implicates RAGE in inflammation and endothelial dysfunction, the role of RAGE in human vascular function and blood flow regulation with advancing age is not well understood. Therefore, this study investigated the relative abundance of RAGE in the SMFAs from young and old humans and the relationship between RAGE abundance and SMFA vasodilatory function.

Methods

RAGE protein expression was assessed in both young (28±4 yrs, n=6) and old (75±7 yrs, n=6) subjects with Western blot analysis. Endothelium‐dependent vasodilatory function was assessed in SMFAs, using pressure myography, in response to flow‐induced shear stress and acetylcholine (ACh). Endothelium‐independent vasodilatory function was assessed, in response to sodium nitroprusside (SNP).

Results

An age‐related attenuation in endothelial‐dependent vasodilatory function was evident in response to flow (Young: 98 ± 15; Old: 30 ± 17%, P < 0.05) and ACh (Young: 98±10; Old: 61±13 %, P < 0.05), while the endothelial‐independent response to SNP was not affected by age. SMFA RAGE expression increased with age (p<0.05) and there was a significant negative correlation between RAGE protein expression and both flow (r2= −0.8783, P < 0.05) and ACh (r2= −0.7824, P < 0.05) induced vasodilation. There was no such relationship for RAGE and SNP induced vasodilation.

Conclusions

These findings imply that RAGE may play a critical role in the age‐related endothelial dysfunction documented in human SMFAs. Furthermore, this RAGE linked endothelial dysfunction may limit skeletal muscle blood flow and contribute to cardiovascular disease in the elderly.

Support or Funding Information

NIH Heart, Lung, Blood Institute grants (HL‐ 091830) and Veterans Affairs Rehabilitation Research and Development Merit (E6910‐R)
Direct Implications for Caveolin‐1 In TRPV1 Mediated NO production: Elucidating A Mechanism Of Vasoreactivity
Authors: Fahmy Joseph N. , Andrei Spencer , DelloStritto Daniel J. , Geldenhuys Werner , Damron Derek S. , Bratz Ian N.
Published: November 2023

Caveolin‐1 (Cav‐1), a scaffolding protein responsible for the formation of caveolae, is ubiquitously expressed and prominently in endothelial cells. Moreover, Cav‐1 is known to associate with several ion channels including various Kv and TRP subtypes. It is well established that when Cav‐1 and endothelial nitric oxide synthase (eNOS) are bound, Cav‐1 exerts an inhibitory effect on eNOS activity and thus decreases NO production until a Ca2+ dependent signal untethers the proteins. Our lab and others have shown that TRPV1 regulates eNOS activity and NO production; specifically, via a calcium‐mediated AKT‐CaMKII‐AMPK signaling cascade. Therefore, we hypothesize that TRPV1, Cav‐1, and eNOS form a regulatory protein complex in endothelial cells responsible for the modulation of NO production. Examination of the TRPV1 amino acid sequence revealed 4 potential Cav‐1 binding motifs (where Ø represents any aromatic amino acid and X represents any amino acid): ØXXØXXXXØØ, ØXXØXXXØ, ØXXXØXXØ, and ØXXXØXØ. Computational modeling predicted an interaction between TRPV1 and Cav‐1. Confirmation of a direct TRPV1/Cav‐1 association was illustrated using Proximity Ligation Assay (PLA) in mouse coronary endothelial cells, which was increased following treatment with the TRPV1 agonist capsaicin. Furthermore, capsaicin treatment decreased the interaction between Cav‐1 and eNOS visualized with PLA, further defining the nature of this NO mediated cellular response. Similarly, NO production increased following capsaicin treatment. In conclusion, this is the first evidence of a direct interaction between TRPV1 and Cav‐1 and its implications in the regulation of NO production and vascular function.
Contribution of Adiponectin to Vascular Responses in Bone Resistance Arteries in Mice
Authors: Ghosh Payal , Hotta Kazuki , Lucero Tiffany , Borodunovich Kyle , Cowan Morgan , Bramy Jeremy , Behnke Bradley , Delp Michael , Delp Judy
Published: November 2023

Adiponectin has been reported to alter structural properties of bone, which may be influenced by altered regulation of bone blood flow and related vasomotor properties in the bone resistance vasculature. Therefore, the purpose of this study was to determine the effects of adiponectin deletion on bone resistance arterial function.

Adult male C57BL/6 wild‐type (WT) and homozygous adiponectin knockout (AdipoKO) mice were obtained for study. Femoral principle nutrient arteries (PNA) were isolated, cannulated and pressurized for in vitro study. Endothelium‐dependent (ACh, 10−9–10−4 M) and ‐independent (Dea‐NONOate, 10−9–10−4 M) vasodilation, and myogenic (0 to 130 cmH2O) responsiveness were evaluated.

Endothelium‐dependent vasodilation was significantly impaired in PNA arteries from AdipoKO mice compared to arteries from WT mice; however, no significant differences in endothelium‐independent vasodilation were observed between arteries from WT and AdipoKO mice. Myogenic responsiveness tended to be greater in arteries from AdipoKO mice than arteries from WT mice. The greater myogenic tone seen in PNA arteries from AdipoKO mice may be linked to endothelial dysfunction.

These results indicate that in bone resistance arteries, adiponectin modulates endothelium‐dependent vasodilation but does not affect vasodilatory function of the vascular smooth muscle. These alterations in vasomotor function could potentially play a role in alterations of bone structural properties associated with changes in circulating adiponectin.
Pharmacological or Genetic Interruption of Focal Adhesion Kinase Improves Endothelial Barrier Dysfunction Resulting from Ischemia/Reperfusion Injury
Authors: Haines Ricci J , Zhang Shimin , Eitnier Rebecca A , Wang Fang , Yang Clement GY , Wu Mack H
Published: November 2023

Ischemia reperfusion (I/R) injury results from reoxygenation following an initial ischemic insult. Although oxygen levels are restored during reperfusion, the abrupt increase in blood supply results in high levels of reactive oxygen species that leads to endothelial barrier dysfunction and exacerbates the tissue injury originally elicited by the ischemic event. One therapeutic strategy used to enhance the endothelial barrier and prevent further tissue damage is to treat the exacerbated tissue damage caused by reperfusion. Our lab and others have shown that focal adhesion kinase (FAK) is a key player that participates in the signaling events required for endothelial barrier dysfunction. Therefore, we hypothesized that targeting FAK pharmacologically would improve I/R injury in vitro and in vivo. Using murine intestinal endothelial cells, we performed electric cell‐substrate impedance sensing (ECIS) assays with and without FAK inhibitor (PF573228, 1μM) in the presence of H2O2 (250μM). Results showed that FAK inhibition significantly attenuated barrier dysfunction induced by H2O2. Similarly, we also utilized FITC‐albumin flux assays to determine whether targeting FAK improved barrier function resulting from H2O2. These experiments showed that the increase of endothelial albumin flux due to treatment with H2O2 was reduced by approximately 50% when cells were pretreated with FAK inhibitor. Western blots of endothelial cell lysates treated with H2O2 indicated that FAK is activated in as little as 10 minutes and remains elevated for 60 minutes of treatment. Other experiments showed that the use of PF573228 dramatically reduced detection of active FAK (pY397 FAK) as well as total tyrosine phosphorylated proteins despite the presence of inflammatory signals. Using an in vivo model of I/R injury and endothelial specific, tamoxifen inducible, FAK knockout transgenic animals, as well as wild type animals given PF573228, we showed that interruption of FAK activity or expression reduced albumin leakage induced by I/R injury by over two‐fold, while neither mean arterial blood pressure nor diameter was altered by the drug or genetic manipulation. Taken together, these results suggest that targeting FAK may be a novel strategy to improve endothelial barrier dysfunction that occurs as a consequence of I/R injury.

Support or Funding Information

NIH HL‐120954
The Role of Formyl Peptide Receptors (FPRs) in Cerebral Microvascular Dysfunction in Sickle Cell Disease
Authors: Gavins Felicity N. E. , Ansari Junaid , Senchenkova Elena Y. , Vital Shantel A.
Published: November 2023

Cerebrovascular disease is a leading cause of death worldwide. It is a serious complication of sickle cell disease (SCD), an inherited autosomal recessive disorder, resulting from a single amino acid substitution in the haemoglobin β chain. Patients with SCD are more susceptible to thrombotic events, in which leukocytes, platelets and erythrocyte adhesion have all been implicated. It is therefore not surprising that SCD is frequently referred to as a “hypercoagulable state” characterized by a proinflammatory and prothrombogenic phenotype.

The causes for the prothrombotic SCD phenotype may relate to a well‐established link between thrombosis and inflammation: Systemic inflammation can beget local thrombosis, and thrombosis can amplify inflammation. Accumulating data linking inflammation and thrombosis supports the hypothesis that anti‐inflammatory mediators may limit thrombosis and that anti‐thrombotic mediators may reduce vascular inflammation. One such anti‐inflammatory endogenous mediator is the glucocorticoid‐regulated protein Annexin A1 (AnxA1), which has been shown to dampen inflammation by e.g. the inhibition of leukocyte recruitment and cytokine release, promotion of apoptosis, stimulation of phagocytosis, and decreasing vascular permeability. The biological effects of AnxA1 occur through an interaction with the formyl peptide receptors (FPRs). In humans three FPRs (FPR1, FPR2, and FPR3) regulate innate inflammatory responses. Their function has been most extensively investigated in the context of leukocyte recruitment and activation, where FPRs promote cell motility (chemotaxis) and microbicidal respiratory burst.

Here we have tested the effect of targeting the AnxA1‐FPR pathway as a therapeutic strategy for SCD. Data presented during the talk adds to the current literature suggesting a pro‐inflammatory and pro‐thrombogenic cerebral microcirculation is found in SCD mice. Furthermore, SCD appears to promote the formation of thrombosis, which can be inhibited by the administration of AnxA1 via the FPR pathway.

Support or Funding Information

The NIH/NHLBI (HL125572‐01A1)