Female and male obese Zucker rats display differential inflammatory mediator and long non-coding RNA profiles.

AIMS: The goal of this study was to identify mediators in peri-lymphatic adipose tissue (PLAT) that are altered in obese versus lean Zucker rats, with focus on potential sex differences MAIN METHODS: Mesenteric PLAT was analyzed with protein and lncRNA arrays. Additional RT-PCR confirmation was performed with epididymal/ovarian fat. KEY FINDINGS: MCP-1, TCK-1, Galectin-1, Galectin-3, and neuropilin-1 were elevated in PLAT from obese rats of both sexes. However, 11 additional proteins were elevated only in obese males while 24 different proteins were elevated in obese females. Profiling of lncRNAs revealed lean males have elevated levels of NEAT1, MALAT1 and GAS5 compared to lean females. NEAT1, MALAT1, and GAS5 were significantly reduced with obesity in males but not in females. Another lncRNA, HOTAIR, was higher in lean females compared to males, and its levels in females were reduced with obesity. Obese rats of both sexes had similar histologic findings of mesenteric macrophage crown-like structures and hepatocyte fat accumulation. SIGNIFICANCE: While obese male and female Zucker rats both have increased inflammation, they have distinct signals. Future studies of the proteome and lncRNA landscape of obese males vs. females in various animal models and in human subjects are warranted to better guide development of therapeutics for obesity-induced inflammation.

Impact of Goreisan components on rat mesenteric collecting lymphatic vessel pumping.

BACKGROUND: Goreisan is a traditional herbal formulation with diuretic properties tested as a clinical therapeutic to alleviate lymphedema in Japan. The present study aimed to determine how Goreisan and its five different components affect lymphatic pump function. METHODS: Mesenteric collecting lymphatics were isolated from anesthetized Sprague-Dawley rats and mounted on resistance-matched glass micropipettes in a 37°C physiological salt solution bath for studies. Diameter was continuously measured to obtain the following lymphatic pump parameters: contraction frequency (CF), end diastolic diameter (EDD), and end systolic diameter (ESD), contraction amplitude (AMP), ejection fraction (EF), and fractional pump flow (FPF). Goreisan and each of its components (Cinnamomi Cortex, Atractylodis Rhizoma, Alismatis Rhizoma, Polyporus, and Poria) were applied to the bath at concentrations of 1-30 µg/mL. RESULTS: The results show that while Goreisan causes no significant changes to lymphatic pumping, Alismatis Rhizoma and Polyporus each significantly reduce CF and FPF. In addition, rats that received oral administration of Goreisan and Alismatis Rhizoma for 1 week had elevated expression of VEGFR-3 in their mesenteric collecting lymphatics. CONCLUSIONS: Collectively, the results suggest that some components of Goreisan have a direct, rapid impact on lymphatic pumping. These findings provide new insights but also raise new questions about the therapeutic potential of Goreisan in patients with secondary lymphedema.

Evidence of functional ryanodine receptors in rat mesenteric collecting lymphatic vessels.

In the current study, the potential contributions of ryanodine receptors (RyRs) to intrinsic pumping and responsiveness to substance P (SP) were investigated in isolated rat mesenteric collecting lymphatic vessels. Responses to SP were characterized in lymphatic vessels in the absence or presence of pretreatment with nifedipine to block L-type Ca2+ channels, caffeine to block normal release and uptake of Ca2+ from the sarcoplasmic reticulum, ryanodine to block all RyR isoforms, or dantrolene to more selectively block RyR1 and RyR3. RyR expression and localization in lymphatics was also assessed by quantitative PCR and immunofluorescence confocal microscopy. The results show that SP normally elicits a significant increase in contraction frequency and a decrease in end-diastolic diameter. In the presence of nifedipine, phasic contractions stop, yet subsequent SP treatment still elicits a strong tonic contraction. Caffeine treatment gradually relaxes lymphatics, causing a loss of phasic contractions, and prevents subsequent SP-induced tonic contraction. Ryanodine also gradually diminishes phasic contractions but without causing vessel relaxation and significantly inhibits the SP-induced tonic contraction. Dantrolene treatment did not significantly impair lymphatic contractions nor the response to SP. The mRNA for all RyR isoforms is detectable in isolated lymphatics. RyR2 and RyR3 proteins are found predominantly in the collecting lymphatic smooth muscle layer. Collectively, the data suggest that SP-induced tonic contraction requires both extracellular Ca2+ plus Ca2+ release from internal stores and that RyRs play a role in the normal contractions and responsiveness to SP of rat mesenteric collecting lymphatics.NEW & NOTEWORTHY The mechanisms that govern contractions of lymphatic vessels remain unclear. Tonic contraction of lymphatic vessels caused by substance P was blocked by caffeine, which prevents normal uptake and release of Ca2+ from internal stores, but not nifedipine, which blocks L-type channel-mediated Ca2+ entry. Ryanodine, which also disrupts normal sarcoplasmic reticulum Ca2+ release and reuptake, significantly inhibited substance P-induced tonic contraction. Ryanodine receptors 2 and 3 were detected within the smooth muscle layer of collecting lymphatic vessels.

Sphingosine-1-Phosphate Reduces Hemorrhagic Shock and Resuscitation-Induced Microvascular Leakage by Protecting Endothelial Mitochondrial Integrity.

Excessive microvascular permeability is a serious complication following hemorrhagic shock and resuscitation (HSR). S1P has been shown to ameliorate microvascular leakage in a model of combined alcohol intoxication and HSR. In the current study, we tested the hypothesis that S1P reduces HSR-induced microvascular leakage by preserving endothelial cell junctional structure and the endothelial glycocalyx through the protection of mitochondrial function. We used an established in vivo rat model of conscious HSR and assessed microvascular leakage, endothelial glycocalyx integrity, and mitochondrial function by intravital microscopy. Junctional integrity in the mesenteric microcirculation was assessed by confocal microscopy. Cultured rat intestinal microvascular endothelial cells monolayers were used to test the ability of S1P to protect against glycocalyx shedding and endothelial barrier dysfunction caused by direct disruption of mitochondrial integrity due to inhibition of mitochondrial complex III. The results show that in vivo, S1P protects against HSR-induced hyperpermeability, preserves the expression of adherens junctional proteins, and protects against glycocalyx degradation. S1P treatment during HSR also protects against mitochondrial membrane depolarization. S1P also protects against mitochondrial dysfunction-induced endothelial barrier dysfunction and glycocalyx degradation by acting through mitochondrial complex III. Taken together, our data indicate that S1P protects against HSR-induced mitochondrial dysfunction in endothelial cells, which in turn improves the structure of the endothelial glycocalyx after HSR and allows for better junctional integrity to the prevention of excess microvascular permeability.

Modulation of mesenteric collecting lymphatic contractions by σ1-receptor activation and nitric oxide production.

Recently, it has been reported that a σ-receptor antagonist could reduce inflammation-induced edema. Lymphatic vessels play an essential role in removing excess interstitial fluid. We tested the hypothesis that activation of σ-receptors would reduce or weaken collecting lymphatic contractions. We used isolated, cannulated rat mesenteric collecting lymphatic vessels to study contractions in response to the σ-receptor agonist afobazole in the absence and presence of different σ-receptor antagonists. We used RT-PCR and Western blot analysis to investigate whether these vessels express the σ1-receptor and immunofluorescence confocal microscopy to examine localization of the σ1-receptor in the collecting lymphatic wall. Using N-nitro-l-arginine methyl ester (l-NAME) pretreatment before afobazole in isolated lymphatics, we tested the role of nitric oxide (NO) signaling. Finally, we used 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence as an indicator to test whether afobazole increases NO release in cultured lymphatic endothelial cells. Our results show that afobazole (50-150 µM) elevated end-systolic diameter and generally reduced pump efficiency and that this response could be partially blocked by the σ1-receptor antagonists BD 1047 and BD 1063 but not by the σ2-receptor antagonist SM-21. σ1-Receptor mRNA and protein were detected in lysates from isolated rat mesenteric collecting lymphatics. Confocal images with anti-σ1-receptor antibody labeling suggested localization in the lymphatic endothelium. Blockade of NO synthases with l-NAME inhibited the effects of afobazole. Finally, afobazole elicited increases in NO production from cultured lymphatic endothelial cells. Our findings suggest that the σ1-receptor limits collecting lymphatic pumping through a NO-dependent mechanism.NEW & NOTEWORTHY Relatively little is known about the mechanisms that govern contractions of lymphatic vessels. σ1-Receptor activation has been shown to reduce the fractional pump flow of isolated rat mesenteric collecting lymphatics. The σ1-receptor was localized mainly in the endothelium, and blockade of nitric oxide synthase inhibited the effects of afobazole.

Demonstration of the rat ischemic skin wound model.

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.