(M-491) Perivascular Adipose Tissue from High Fat Diet fed Rats Impairs Endothelial-Dependent Vasodilation

Obesity is a strong risk factor for the development of cardiovascular disease (CVD), the leading cause of worldwide mortality. CVD is associated with endothelial dysfunction, defined as increased inflammation and decreased nitric oxide production. Lifestyle and behavioral choices, such as a high fat diet (HFD) and obesity, increase the risk of CVD. For example, a HFD leads to inflamed perivascular adipose tissue (PVAT) surrounding blood vessels, which exacerbates endothelial dysfunction (1). Moreover, women have a higher propensity for obesity than men, which increases their CVD risk (2). However, there is a lack of CVD studies investigating how PVAT affects endothelial function in obese women. We hypothesize that female rats fed a HFD will have higher endothelial inflammation and lower nitric oxide production compared to male rats fed a HFD, thus impairing their vascular function. To evaluate this hypothesis, we used pressure myography to measure vascular function, western blots for protein analysis, and quantitative polymerase chain reaction (qPCR) for gene expression analysis in male and female rats fed a HFD. 

22-week-old male and female Sprague-Dawley rats (n=6/sex) were fed a normal chow or HFD for 16 weeks. The rats were euthanized and PVAT was removed from the thoracic artery and cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 1% penicillin-streptomycin for 24 hours (40 mg PVAT/250 µL DMEM). Endothelial-dependent vasodilation was measured by pressurizing rat mesenteric arteries, treating with PVAT conditioned media (PVAT-CM) or DMEM for 1 hour, and running an acetylcholine dose response. The half maximal acetylcholine vasodilation response was quantified as the pEC50. We then studied how PVAT-CM affects endothelial function by treating rat aortic endothelial cells (RAEC) in endothelial cell growth media supplemented with 5% fetal bovine serum and treated with 4 µM yoda1 ± 10% PVAT-CM. The piezo1-agonist yoda1 facilitates endothelial nitric oxide synthase phosphorylation (p-eNOS) to generate nitric oxide. Protein changes were measured by Western blot. RAECs were lysed in RIPA buffer and proteins were separated using 4-12% Bis-Tris gels then transferred to PVDF membranes and probed for p-eNOS, eNOS, and GAPDH. Endothelial inflammation was measured using white blood cell (WBC) adhesion. WBC were isolated using an Acrodisc WBC filter and labeled with calcein green. We then imaged samples after incubating the RAECs with 2x106 WBCs/mL for 10 minutes. Finally, we measured gene expression changes with qPCR. RNA was isolated using an RNEasy Kit. Reverse transcription was run using a cDNA Reverse Transcription Kit, and qPCR was run with Taqman primers for eNOS gene NOS3, inflammatory gene IL6, and housekeeper HPRT1. 

We first performed pressure myography to determine how rat PVAT-CM affects vascular function. Our acetylcholine dose-response experiments demonstrated that PVAT-CM from HFD female rats decreased vasodilation in sex- and diet-matched mesenteric arteries (pEC50 = 6.66 ± 0.18) compared to those treated with DMEM (pEC50 = 6.93 ± 0.13; p = 0.0323) (Figure 1a). We then investigated how PVAT-CM impacted RAEC eNOS phosphorylation by Western blot. Neither female nor male HFD PVAT-CM decreased RAECs p-eNOS (Figure 1b), suggesting that HFD PVAT-CM does not impair endothelial nitric oxide via eNOS phosphorylation. We then studied how PVAT-CM affected RAEC inflammation. PVAT-CM from both male and female HFD rats increased RAEC WBC adhesion, and female PVAT-CM may induce a higher inflammatory response (WBC= 59.33 ± 5.64) than the male PVAT-CM (WBC=  48.53 ± 15.39) (Figure 1c). Finally, we investigated whether female and male PVAT-CM altered RAEC eNOS and inflammatory gene expression. qPCR showed no significant changes in NOS3 (eNOS) or the inflammatory mediator interleukin-6 (IL6) in cells treated with PVAT-CM from either female or male rats compared to untreated RAECs (data not shown). 

Overall, our results showed that PVAT-CM from female rats fed a HFD impaired vasodilation, but these effects were not seen with PVAT-CM from male rats fed a HFD. Moreover, these effects were not caused by impaired eNOS phosphorylation or inflammatory gene expression, though inflammatory response may have been higher in RAECs treated with PVAT-CM from female HFD rats. Future studies include measuring other factors that reduce nitric oxide availability, such as reactive oxygen species.

Funded was provided to D.D.T through the UMD ADAPT program, C.M.W through the NSF Graduate Research Fellowship (DGE 180340), G.S.S through the AHA Postdoctoral Fellowship (916512), and A.M.C. by NIH (R21EB028466) and NSF (CMMI 1916814). 

[1]    Kajikawa, M., Biomedicines, (2022a). 10(7): 1745. 

[2]    Manrique-Acevedo., et al. International Journal of Obesity, 44(6)(2021):1210–1226.