Seven days of statin treatment improves nitric-oxide mediated endothelial-dependent cutaneous microvascular function in women with endometriosis

The Institutional Review Board at The Pennsylvania State University approved all experimental procedures and protocols. A Food and Drug Administration Investigational Drug Number was obtained for all protocols (IND 78,954). Verbal and written informed consent were voluntarily obtained from all participants before participation and in accordance with the guidelines set forth by the Declaration of Helsinki. All participants were screened by clinical staff. Screening included a physical examination, medical health history questionnaire, and a blood chemistry analysis (Chem 24, Quest Diagnostics, Pittsburgh, PA). All participants were free from stage II hypertension, as well as renal, pulmonary, neurological, or dermatological diseases, and had never experienced an adverse cardiovascular disease event. Participants did not use tobacco products. One woman with endometriosis was taking a selective serotonin reuptake inhibitor. All other participants were not taking over-the-counter or prescription medications with primary or secondary cardiovascular effects (e.g., antihypertensives, hormonal therapy, statins, anticoagulants, antidepressants) at the time of testing. All women were premenopausal. Healthy control participants had regularly occurring menstrual cycles (28–32 days, Table 1 ) but we did not control for menstrual cycle phase during the study visit ( 26 ). All endometriosis patients had a confirmed diagnosis prior to enrollment via laparoscopy. All participants underwent a cutaneous microdialysis experiment to assess microvascular endothelial function. Participants were instructed to abstain from caffeine and alcohol for 12 hours and strenuous physical activity for 24 hours prior to the experimental visit. Two intradermal microdialysis fibers (10mm, 55kDa, CMA Linear 31 probe, Harvard Apparatus, Holliston, MA) were inserted into the ventral forearm skin for the local delivery of either lactated Ringer’s solution alone (Control), or with Ringer’s solution and 0.015 M N G -nitro- l -arginine methyl ester ( l -NAME; Calbiochem, EMD Millipore, Billerica, MA) to non-selectively inhibit NO synthase. In endometriosis patients only, a third intradermal microdialysis fiber was placed for the local delivery of 0.00002 M atorvastatin (statin; USP, Rockville, MD). After microdialysis fiber insertion, 60–90 min were allowed for hyperemia resolution during which site-specific pharmacological agents were perfused (2 μL/min; Hive controller and microinfusion pumps; BASi, West Lafayette, IN). Cutaneous red blood cell flux was continuously measured directly over each microdialysis site with an integrated laser-Doppler flowmeter probe placed in a local heating unit (VP12 and VHP2; Moor Instruments, Wilmington, DE) set to a thermoneutral 33°C. After baseline measurements, progressively increasing concentrations of the endothelium-dependent agonist acetylcholine (ACh; 10 −10 to 10 −1 M; USP, Rockville, MD) were co-perfused with lactated Ringer’s, l -NAME, or statin (endometriosis patients only) sequentially for 5 min each. At the conclusion of the dose-response protocols, 0.028 M sodium nitroprusside (USP) was perfused and the local skin temperature was increased to 43°C to elicit maximal cutaneous vasodilation ( 27 – 30 ). Automated brachial blood pressure (Connex Spot Monitor, Welch Allyn, Skaneateles Falls, NY) was measured every 5 min throughout the protocol. A subset (n=6) of endometriosis patients participated in a seven-day oral atorvastatin (Lipitor®) therapy (10mg/day). These subjects returned to the laboratory on day 7, having taken the final dose that morning. We repeated the microdialysis experimental visit as described above. Intradermal microdialysis data collection procedures and the pharmacological efficacy of the site-specific inhibitors have been previously reported ( 27 – 32 ). Data were recorded at 1000 Hz and stored for offline analysis (Powerlab and LabChart, ADInstruments, Bella Vista, NSW, Australia). Average values for red cell flux (perfusion units) were obtained during 5 min of baseline, during the last minute of each dose, and at maximum. Cutaneous vascular conductance (CVC) was calculated as red cell flux divided by mean arterial pressure. Due to the heterogeneity of capillary density at each microdialysis site, CVC was normalized as a percentage of the site-specific maximum (CVC %max ), which is a standard normalization procedure for data derived from laser-Doppler flowmetry ( 27 – 32 ). Area under the dose-response curve (AUC) was calculated using the trapezoid rule (Prism v8.1, GraphPad Software, La Jolla, CA). The NO-dependent contributions were calculated as the difference between the AUC of the control site and the L-NAME sites ( 27 , 28 , 33 ). All data were analyzed with either a one- or two-way repeated measures ANOVA (Prism v8.1, GraphPad Software, La Jolla, CA; IBM Corp. Released 2019. IBM SPSS Statistics for Windows, v. 26.0. Armonk, NY: IBM Corp; SAS v. 9.4; Cary, NC). When appropriate, post hoc Tukey–Kramer corrections were applied to account for multiple comparisons. Linear regression analyses were performed using a Pearson’s correlation coefficient. Significance was set a priori at α < 0.05. Text and table results are presented as mean ± SD. Figure results are presented as means ± SE for visual clarity of main effects. Assuming an effect size of d = 0.68 ( 27 ), we determined a priori (power = 0.80, α = 0.05) a sample size of n = 8 would be sufficient to detect a significant difference in microvascular function between groups (healthy control vs. endometriosis patients). Based on previous data ( 28 ), assuming an effect size of d = 0.65 we determined a priori (power = 0.80, α = 0.05) a sample size of n = 8 would be sufficient to determine a significant difference between pre- and post-intervention (7-day systemic statin treatment). We originally recruited 8 women with endometriosis to participate in the intervention; however, 2 participants dropped out (n=1 developed COVID-19, n=1 received surgical treatment for endometriosis). However, the effects pre- to post-intervention were large (NO contribution: d= 1.49), despite 2 drop-outs.

A total of 8 endometriosis patients and 8 age- and body mass index-matched women (controls) without endometriosis participated in the study. There were no differences in characteristics between groups ( Table 1 ). In the control group, 6 were normotensive, 1 had elevated blood pressure (122/78 mmHg), and 1 had stage 1 hypertension (124/86 mmHg). In the endometriosis group, 6 were normotensive, and 2 had stage 1 hypertension (120/81 and 135/88 mmHg) according to the 2017 AHA/ACC guidelines ( 34 ). With the exceptions of 1 endometriosis patient who had hypercholesterolemia (LDL: 155mg/dl) and was obese (body mass index = 36 kg/m 2 ), all women were otherwise heathy ( Table 1 ). A breakdown of medications and menstrual cycle status is presented in Table 1 . ACh-induced vasodilation was attenuated in the endometriosis group ( Figure 1 , main effect p=0.03). In controls the local co-perfusion of L-NAME decreased the vasodilator response to ACh at the highest six doses of ACh ( Figure 1 , all p<0.01). In women with endometriosis, the vasodilatory response was only attenuated at the highest dose of ACh ( Figure 1 , p<0.01). The NO-mediated contribution to ACh-induced vasodilation was greater in the control group compared to the endometriosis group (217±120 vs. 88±97 AU, p=0.03). 6 of the 8 women with endometriosis completed the seven-day systemic atorvastatin intervention. Seven days of systemic atorvastatin intervention lowered total cholesterol ( Figure 2A , p<0.01) and low-density lipoprotein cholesterol ( Figure 2C , p<0.01); but did not impact high-density lipoprotein cholesterol ( Figure 2B , p=0.43) or mean arterial pressure (84±13 vs. 83±10 mmHg, p=0.76). The oral statin treatment increased the ACh-induced vasodilatory response ( Figure 3A ; main effect p<0.01). After the oral statin treatment, the local co-perfusion of L-NAME attenuated the ACh-induced response ( Figure 3A , p <0.01). The NO-mediated contribution increased pre- to post-intervention ( Figure 3B , p=0.05). Prior to oral statin treatment, local statin co-perfusion (intradermal microdialysis) augmented ACh-induced vasodilation ( Figure 4A , p<0.01). After statin treatment, local statin co-perfusion attenuated ACh-induced vasodilation ( Figure 4B , p=0.01). There were no associations between the change in cholesterol and the change in vasodilatory function (all p>0.05).

The results of this proof-of-concept trial demonstrate that women with endometriosis have impaired endothelial-dependent microvascular function, mediated in part through reduced NO-dependent mechanisms. Localized statin perfusion directly to the cutaneous microvasculature prior to systemic statin treatment modestly increased microvascular endothelial-dependent vasodilation. Seven days of oral statin improved endothelium-dependent dilation and NO-dependent vasodilation. These data suggest that short term oral statin treatment improved microvascular endothelium-dependent vasodilation and that the local impact of statin also increased microvascular function potentially through anti-inflammatory, anti-oxidant, or through the acute inhibition of the lectin-like oxidized LDL (LOX-1) receptor mechanisms ( 20 – 22 , 35 – 37 ). These data provide compelling support for future randomized clinical trials investigating statin therapy interventions for the treatment of endometriosis-associated microvascular dysfunction. Santoro et al., (2012) observed ~4.6% lower flow-mediated vasodilation (FMD) in women with endometriosis compared to healthy controls; however, no differences in carotid intima-media thickness were observed between groups suggesting that women with endometriosis have impaired conduit artery vascular function despite no alterations in vascular structure ( 17 ). Impaired endothelial function is indicative of early changes in the atherosclerotic disease process putatively mediated by chronic systemic inflammation in women with endometriosis ( 3 , 8 , 17 ). Interestingly, FMD was improved in women with endometriosis 2 years after surgical excision of endometrial lesions such that there was no longer a difference between healthy controls and women with endometriosis ( 16 ). FMD is a gold standard for assessing conduit artery vasodilator capacity to a shear stimulus. The underlying mechanisms of FMD include both endothelial and vascular smooth muscle pathways, as well as NO-mediated and non-NO-mediated mechanisms (e.g. endothelial derived hyperpolarizing factors, prostaglandins) ( 38 ). In the present study we used the cutaneous circulation to interrogate endothelial function in the microcirculation where decrements in vascular function often precede or evolve in parallel with conduit artery endothelial function ( 13 , 14 ). Using this approach, we pharmaco-dissected the NO-mediated endothelial dependent vasodilatory pathway using the endothelium-dependent agonist acetylcholine paired with direct nitric oxide synthase (NOS) blockade. Our results indicate that women with endometriosis display impaired microvascular endothelial function mediated, in part, by reductions in NO bioavailability. Our results are novel because we demonstrate reductions in NO-dependent mechanisms in the microcirculation where previous studies have inferred these results based on indirect measures in conduit arteries. Statins are a cornerstone treatment for the management of atherosclerotic disease risk. We have demonstrated that statins are effective at improving microvascular NO-dependent endothelial function in middle-aged adults with dyslipidemia ( 23 – 25 ). In the current study, seven days of low-dose oral statins improved microvascular function in women with endometriosis. Importantly, the majority of the subject cohort did not have clinically significant hypercholesterolemia. One participant had elevated cholesterol, but removing this participant from the analysis did not change the overall result. We had originally designed the study postulating that plasma cholesterol concentrations would not significantly change after this short-term treatment of the lowest dose of atorvastatin. While there was no relation between the change in cholesterol and changes in microvascular function, we cannot exclude the possibility that reductions in plasma lipids may have impacted our results. Statins mediate their beneficial effects on the vasculature in a number of different ways including directly reducing cholesterol synthesis, and indirectly through exerting non-specific antioxidant and anti-inflammatory effect ( 20 – 22 , 35 , 36 ), and antagonizing the LOX-1 receptor ( 20 , 37 ). LOX-1 is a ubiquitously expressed scavenger receptor that is directly agonized by ox-LDL and upregulated by inflammatory cytokines that are known to be elevated in women with endometriosis ( 39 ). LOX-1 is an upstream signaling initiator that promotes increased oxidant production, and reduced NO metabolism ( 39 ) resulting in pronounced endothelial dysfunction. Our results suggest that LOX-1 is a potential target mediating endothelial dysfunction in women with endometriosis. While the pleotropic effects of lipophilic statins are well documented in cohorts with traditional cardiovascular disease risk factors ( 36 ), their precise mechanisms in those with non-traditional cardiovascular disease risk including endometriosis have not been explored. Interestingly, statins decrease endometrial lesional load in animal models of endometriosis through modulating the expression of genes encoding for estrogen receptors and decreasing inflammation ( 19 ). In order to examine the direct of effects of statins on the microcirculation, thus eliminating the systemic effect such as reducing cholesterol, we locally perfused atorvastatin via intradermal microdialysis. The local infusion of atorvastatin moderately augmented the vasodilatory responses to the endothelium-dependent agonist acetylcholine. We postulate that this improvement in endothelial microvascular function is through antioxidant mechanisms and/or attributable to direct LOX-1 antagonism. Statins inhibit LOX-1 receptor activation through direct steric hindrance properties and induce destabilization of the dimerized LOX-1 receptor ( 20 , 37 ). It was hypothesized that the addition of the local statin infusion after oral treatment would result in a ceiling effect, such that no additional improvement in microvascular function would be observed. However, after seven days of oral statins, the addition of localized statin modestly attenuated the endothelial-dependent vasodilation. We speculate that this may be due to the high antioxidant load, which can induce variable responses, including reductions in NO-dependent vasodilation in healthy subjects ( 40 – 43 ). While endometriosis is a local inflammatory syndrome, the inflammatory process is systemic ( 3 ) and underlies many of the co-morbidities associated with this devastating disease. Endometriosis and atherosclerotic CVD are both inflammation-induced diseases ( 8 ). Robust epidemiologic data demonstrate a clear association between endometriosis, inflammation and CVD risk ( 5 , 9 – 11 , 44 ), the leading cause of death in women worldwide ( 9 ). Investigations into the mechanistic links and putative therapies are necessary for reducing cardiovascular disease risk in women with endometriosis. Statins have been shown to effectively reduce endometriotic lesions in primate and murine models ( 3 , 18 , 19 ). Statins have also been suggested for off label use to reduce systemic inflammation and estrogen receptor expression after excision surgery ( 45 ). Lowering cholesterol through dietary manipulation is also recommended for endometriosis patients to relieve endometriosis-related symptoms ( 46 ), although direct mechanistic evidence is lacking. Randomized control trials are needed to investigate statins as a component for the management of endometriosis including the long term mitigation of elevated CVD risk in women with endometriosis. As a proof-of-concept-study, the small sample size is a limitation to the interpretation and translatability of the results. However, our repeated measures design provided an appropriately statistically powered approach for determining the impact of oral statin treatment on microvascular function in women with endometriosis. Several participants had elevated and stage one hypertension according to the 2017 AHA/ACC guidelines ( 34 ). This elevated CVD risk profile is common in women with endometriosis in this age range ( 9 ). We have previously observed no decrements in endothelial-dependent microvascular function in those with stage 1 HTN ( 27 ). Nevertheless, the severity of endothelial dysfunction in this group of women with endometriosis indicates a clear risk for future atherosclerotic CVD, as documented in the epidemiological data ( 13 , 14 ). Finally, we did not control for phase of the menstrual cycle because this was a small proof-of-concept study involving short term statin therapy. In naturally menstruating women, hormone exposure would change over the course of 7 days. Future randomized control trials should aim to control for reproductive hormone exposure. Interestingly, in the present study we observed consistent results among participants, despite the differences of experiment timing, suggesting menstrual cycle phase may not have had a large impact on the current results. In conclusion, the results of this initial proof-of-concept study suggest that short term systemic treatment with a statin improved cutaneous microvascular endothelium-dependent vasodilation in women with endometriosis, via an augmentation of NO-dependent pathways. As such, intervention strategies specifically targeting lowering of cholesterol, reducing systemic inflammation and/or LOX-1 receptor inhibition should be considered for mitigating vascular dysfunction in women with endometriosis.

Endometriosis derives from the presence of endometrium-like tissue in sites outside the uterine cavity. This estrogen-dependent disorder affects up to 1 in 10 women of reproductive age and is associated with chronic pelvic pain and persistent systemic inflammation ( 1 – 8 ). Cardiovascular disease (CVD) is the leading cause of death in women ( 9 ). Epidemiologic data demonstrate a clear association between endometriosis and accelerated CVD risk ( 5 , 9 – 11 ). The chronic, systemic inflammation associated with endometriosis appears to impact cardiovascular function though several mechanisms, leading to vascular endothelial cell dysfunction ( 12 ). Endothelial dysfunction may be the first functional manifestation of atherosclerosis and a primary causative event in the development of overt CVD ( 13 , 14 ). Endothelial dysfunction is also predictive of long-term morbidity and mortality ( 15 ). Otherwise healthy women with endometriosis have impaired conduit artery endothelial function, but the precise mechanisms mediating this are unresolved ( 16 , 17 ). 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been identified as a potential treatment for endometriosis ( 3 , 18 , 19 ). In both murine and primate models of endometriosis, treatment with simvastatin reduces the number and size of endometriotic lesions, and exerts potent systemic anti-inflammatory effects ( 3 , 18 , 19 ). A large body of clinical literature demonstrates that statins have cholesterol-independent effects on the vasculature and improve endothelial NO bioavailability through a variety of mechanisms ( 20 – 22 ). Our published data demonstrate that low dose statin treatment improves endothelial function by improving eNOS coupling in the microvasculature of patients with hypercholesterolemia ( 23 – 25 ). However, the impact of statin treatment on endothelial function in otherwise healthy women with endometriosis is unknown. Given the elevated CVD risk associated with endometriosis and the potential role for statin therapy in improving CVD and endometriosis outcomes, in a proof of concept study we sought to: (1) examine microvascular endothelial dysfunction in women with endometriosis and (2) determine if short-term statin therapy was effective at improving microvascular endothelial-dependent vasodilation in these women. We hypothesized that women with endometriosis would have impaired microvascular endothelial function mediated by a reduction in NO-dependent vasodilation and that short-term statin therapy would improve endothelial function. Using the cutaneous microcirculation, we pharmacologically assessed endothelial function in the cutaneous microcirculation, which is thought to be a validated bioassay for systemic microvascular dysfunction and has been used to examine the mechanisms underlying microvascular dysfunction in multiple pathologies ( 8 , 12 , 23 , 29 , 51 ).