Recently we observed that endothelial cells cultured in tightly confluent monolayers display frequent local lamellipodia, and that thrombin, an agent that increases endothelial permeability, reduces lamellipodia protrusions. This led us to test the hypothesis that local lamellipodia contribute to endothelial barrier function. Movements of subcellular structures containing GFP-actin or VE-cadherin-GFP expressed in endothelial cells were recorded using time-lapse microscopy. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function. Changes in both lamellipodia dynamics and TER were assessed during baseline and after cells were treated with either the barrier-disrupting agent thrombin, or the barrier-stabilizing agent sphingosine-1-phosphate (S1P). The myosin II inhibitor blebbistatin was used to selectively block lamellipodia formation, and was used to test their role in the barrier function of endothelial cell monolayers and isolated, perfused rat mesenteric venules. Myosin light chain (MLC) phosphorylation was assessed by immunofluorescence microscopy. Rac1 and RhoA activation were evaluated using G-LISA assays. The role of Rac1 was tested with the specific inhibitor NSC23766 or by expressing wild-type or dominant negative GFP-Rac1. The results show that thrombin rapidly decreased both TER and the lamellipodia protrusion frequency. S1P rapidly increased TER in association with increased protrusion frequency. Blebbistatin nearly abolished local lamellipodia protrusions while cortical actin fibers and stress fibers remained intact. Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules. Both thrombin and S1P increased MLC phosphorylation and activation of RhoA. However, thrombin and S1P had differential impacts on Rac1, correlating with the changes in TER and lamellipodia protrusion frequency. Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity. Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.

Background Little is known about the impact of type 2 diabetes mellitus (DM) on coronary arteriole remodeling. The aim of this study was to determine the mechanisms that underlie coronary arteriole structural remodeling in type 2 diabetic (db/db) mice. Methods and Results Passive structural properties of septal coronary arterioles isolated from 12- and 16-wk-old diabetic db/db and control mice were assessed by pressure myography. Coronary arterioles from 12-wk-old db/db mice were structurally similar to age-matched controls. By 16-wks of age, coronary wall thickness was increased in db/db arterioles (p < 0.01), while luminal diameter was reduced (Control: 118±5μm; db/db: 102±4μm, p < 0.05), augmenting the wall-to-lumen ratio by 58% (Control: 5.9±0.6; db/db: 9.5±0.4, p < 0.001). Inward hypertrophic remodeling was accompanied by a 56% decrease in elastic modulus (p < 0.05, indicating decreased vessel coronary wall stiffness) and a ~30% reduction in coronary flow reserve in diabetic mice. Interestingly, aortic pulse wave velocity and femoral artery incremental modulus were increased (p < 0.05) in db/db mice, indicating macrovascular stiffness. Molecular tissue analysis revealed increased elastin-to-collagen ratio in diabetic coronaries when compared to control and a decrease in the same ratio in the diabetic aortas. Conclusions These data show that coronary arterioles isolated from type 2 diabetic mice undergo inward hypertrophic remodeling associated with decreased stiffness and increased elastin-to-collagen ratio which results in a decreased coronary flow reserve. This study suggests that coronary microvessels undergo a different pattern of remodeling from macrovessels in type 2 DM.

Alcohol abuse; the most common and costly form of drug abuse, is a major contributing factor to many disease categories. The alcohol-attributable disease burden is closely related to the average volume of alcohol consumption, with dose-dependent relationships between amount and duration of alcohol consumption and the incidence of diabetes mellitus, hypertension, cardiovascular disease, stroke, and pneumonia. The frequent occurrence of alcohol use disorders in the adult population and the significant and widespread detrimental organ system effects highlight the importance of recognizing and further investigating the pathophysiological mechanisms underlying alcohol-induced tissue and organ injury.

Background/ObjectivesExpanding visceral adiposity is associated with increased inflammation and increased risk for developing obesity-related comorbidities. The goal of this study was to examine high fat diet (HFD)-induced differences in adipocyte size and cytokine/chemokine expression in visceral and subcutaneous adipose depots in obesity-prone (OP) and obesity-resistant (OR) rats.MethodsOP and OR rats were fed either a low fat diet (LFD, 10% kilocalories from fat) or HFD (60% kilocalories from fat) for 7 weeks. Adipocyte size and the presence of crown-like structures in epididymal and inguinal adipose tissue were determined. A multiplex cytokine/chemokine panel was used to assess the expression of inflammatory markers in epididymal and inguinal adipose tissues.ResultsA higher percentage of large adipocytes (> 5000 μm2) was detected in the epididymal and inguinal adipose tissues of OP rats and a higher percentage of small adipocytes (< 4000 μm2) was detected in the epididymal and inguinal adipose tissues of OR rats. More crown-like structures were identified in epididymal adipose tissue of OP rats fed a LFD, compared to OR rats. Consumption of a HFD increased the number of crown-like structures in OR, but not OP rats. Epididymal expression of pro-inflammatory cytokines (IL-1β, TNF-α) was higher in OP rats, compared to OR rats fed LFD. HFD consumption increased epididymal expression of GM-CSF, IL-1α, IL-1β, IL-6, MIP-2, and TNF-α in OP and OR rats. Inguinal expression of pro-inflammatory cytokines (IL-1α, IL-1β, TNF-α) was higher in OP rats, compared to OR rats.ConclusionsOverall, these data suggest that a higher susceptibility to developing obesity is characterized by large adipocytes and increased visceral adipose inflammation. Interestingly, in OR rats, the detrimental effects of HFD consumption on visceral adipose inflammation are evident with only small increases in weight and adiposity, suggesting that HFD also increases the risk for obesity-related comorbidities in OR rats.

Objective Acute alcohol intoxication increases intestinal lymph flow by unknown mechanisms, potentially impacting mucosal immunity. We tested the hypothesis that enhanced intrinsic pump function of mesenteric lymphatics contributes to increased intestinal lymph flow during alcohol intoxication. Methods Acute alcohol intoxication was produced by intragastric administration of 30% alcohol to concious, unrestrained rats through surgically-implanted catheters. Time-matched controls received either no bolus, vehicle, or isocaloric dextrose. Thirty minutes after alcohol administration, rats were anesthetized and mesenteric collecting lymphatics were isolated and cannulated to study intrinsic pumping parameters. In separate experiments, mesenteric lymphatics were isolated to examine direct effects of alcohol on intrinsic pump activity. Results Lymphatics isolated from alcohol-intoxicated animals displayed slgnificantly decreased contraction frequency (CF) than the dextrose group, elevated stroke volume index (SVI) versus all other groups, and decreased myogenic responsiveness compared to sham. Elevating pressure from 2 to 4 cm H2O increased the volume flow index 2.4-fold in the alcohol group versus 1.4-fold for shams. Isolated lymphatics exposed to 20 mM alcohol had reduced myogenic tone, without changes in CF or SVI. Conclusions Alcohol intoxication enhances intrinsic pumping by mesenteric collecting lymphatics. Alcohol directly decreases lymphatic myogenic tone, but effects on phasic contractions occur by an unidentified mechanism.

ObjectiveResistance vessel remodeling is controlled by myriad of hemodynamic and neurohormonal factors. This study characterized structural and molecular remodeling in mesenteric resistance arteries (MRAs) in diabetic (db/db) and control (Db/db) mice.MethodsStructural properties were assessed in isolated MRAs from 12 and 16 wk-old db/db and Db/db mice by pressure myography. Matrix regulatory proteins were measured by Western blot analysis. Mean arterial pressure and superior mesenteric blood flow were measured in 12 wk-old mice by telemetry and a Doppler flow nanoprobe, respectively.ResultsBlood pressure was similar between groups. Lumen diameter and medial cross-sectional area were significantly increased in 16 wk-old db/db MRA compared to control, indicating outward hypertrophic remodeling. Moreover, wall stress and cross-sectional compliance were significantly larger in diabetic arteries. These remodeling indices were associated with increased expression of matrix regulatory proteins matrix metalloproteinase (MMP)-9, MMP-12, tissue inhibitors of matrix metalloproteinase (TIMP)-1, TIMP-2, and plasminogen activator inhibitor-1 (PAI-1) in db/db arteries. Finally, superior mesenteric artery blood flow was increased by 46% in 12 wk-old db/db mice, a finding that preceded mesenteric resistance artery remodeling.ConclusionsThese data suggest that flow-induced hemodynamic changes may supersede the local neurohormonal and metabolic milieu to culminate in hypertrophic outward remodeling of type 2 DM mesenteric resistance arteries.

Objective This study investigated the roles of the H1 and H2 histamine receptors, nitric oxide (NO) synthase, and soluble guanylate (sGC) cyclase in histamine-induced modulation of rat mesenteric collecting lymphatic pumping. Methods Isolated rat mesenteric collecting lymphatics were treated with 1–100 μM histamine. Histamine receptors were blocked with either the H1 antagonist mepyramine or the H2 antagonist cimetidine. The role of NO/sGC signaling was tested using the arginine analog L-NAME, the sGC inhibitor ODQ, and sodium nitroprusside (SNP) as a positive control. Results Histamine applied at 100 μM decreased tone and contraction frequency (CF) of isolated rat mesenteric collecting lymphatics. Pharmacologic blockade of either H1 or H2 histamine receptors significantly inhibited the response to histamine. Pretreatment with ODQ, but not L-NAME, completely inhibited the histamine-induced decrease in tone. ODQ pretreatment also significantly inhibited SNP-induced lymphatic relaxation. Conclusions H1 and H2 histamine receptors are both involved in histamine-induced relaxation of rat mesenteric collecting lymphatics. NO synthesis does not appear to contribute to the histamine-induced response. However, sGC is critical for the histamine-induced decrease in tone and contributes to the drop in CF.

As literature indicates, historic racism and implicit bias throughout academia have been profound metrics leading to a lack of diversity, as related to people from underrepresented groups according to race and ethnicity, among biomedical sciences graduate students in U.S. universities. Recognizing such challenges, a team of biomedical scientists and inclusivity educators developed and implemented a pilot training program within an academic health sciences center as an initial step to educate faculty and staff regarding their roles in the promotion of an inclusive academic environment, receptive to all students, including underrepresented students. The 3-h workshop included didactic modules, videos, teaching modules, and active attendee participation. Faculty and staff were presented common terminology and ways to promote the development of an inclusive and diverse academic workforce. Compared with pre-workshop, post-workshop survey results indicated a statistically significant improvement in attendee knowledge of correctly identifying definitions of “implicit bias,” “status leveling,” “color-blind racial attitudes,” “tokenism,” and “failure to differentiate.” Additionally, by the end of the workshop, participants had a statistically significant increase in self-perceptions regarding the importance of improving diversity and recognizing biases and stereotypes in graduate education, knowing what to say when interacting with people from different cultures, and the ability to acknowledge bias when mentoring students from groups underrepresented in the biomedical field. This preliminary initiative was successful in the introduction of faculty and staff to the importance of fostering an inclusive academic environment and thereby developing a diverse workforce.