Lipedema is a painful loose connective tissue disorder characterized by a bilaterally symmetrical fat deposition in the lower extremities. The goal of this study was to characterize the adipose-derived stem cells (ASCs) of healthy and lipedema patients by the expression of stemness markers and the adipogenic and osteogenic differentiation potential. Forty patients, 20 healthy and 20 with lipedema, participated in this study. The stromal vascular fraction (SVF) was obtained from subcutaneous thigh (SVF-T) and abdomen (SVF-A) fat and plated for ASCs characterization. The data show a similar expression of mesenchymal markers, a significant increase in colonies (p < 0.05) and no change in the proliferation rate in ASCs isolated from the SVF-T or SVF-A of lipedema patients compared with healthy patients. The leptin gene expression was significantly increased in lipedema adipocytes differentiated from ASCs-T (p = 0.04) and the PPAR-γ expression was significantly increased in lipedema adipocytes differentiated from ASCs-A (p = 0.03) compared to the corresponding cells from healthy patients. No significant changes in the expression of genes associated with inflammation were detected in lipedema ASCs or differentiated adipocytes. These results suggest that lipedema ASCs isolated from SVF-T and SVF-A have a higher adipogenic differentiation potential compared to healthy ASCs.

The growth and differentiation of adipose tissue-derived stem cells (ASCs) is stimulated and regulated by the adipose tissue (AT) microenvironment. In lipedema, both inflammation and hypoxia influence the expansion and differentiation of ASCs, resulting in hypertrophic adipocytes and deposition of collagen, a primary component of the extracellular matrix (ECM). The goal of this study was to characterize the adipogenic differentiation potential and assess the levels of expression of ECM-remodeling markers in 3D spheroids derived from ASCs isolated from both lipedema and healthy individuals. The data showed an increase in the expression of the adipogenic genes (ADIPOQ, LPL, PPAR-γ and Glut4), a decrease in matrix metalloproteinases (MMP2, 9 and 11), with no significant changes in the expression of ECM markers (collagen and fibronectin), or integrin A5 in 3D differentiated lipedema spheroids as compared to healthy spheroids. In addition, no statistically significant changes in the levels of expression of inflammatory genes were detected in any of the samples. However, immunofluorescence staining showed a decrease in fibronectin and increase in laminin and Collagen VI expression in the 3D differentiated spheroids in both groups. The use of 3D ASC spheroids provide a functional model to study the cellular and molecular characteristics of lipedema AT.

Aging is a nonmodifiable risk factor for cardiovascular disease associated with arterial stiffening and endothelial dysfunction. We hypothesized that sex differences exist in vascular aging processes and would be attenuated by global deletion of the G protein-coupled estrogen receptor. Blood pressure was measured by tail cuff plethysmography, pulse wave velocity (PWV) and echocardiography were assessed with high resolution ultrasound, and small vessel reactivity was measured using wire myography in adult (25 weeks) and middle-aged (57 weeks) male and female mice. Adult female mice displayed lower blood pressure and PWV, but this sex difference was absent in middle-aged mice. Aging significantly increased PWV but not blood pressure in both sexes. Adult female carotids were more distensible than males, but this sex difference was lost during aging. Acetylcholine-induced relaxation was greater in female than male mice at both ages, and only males showed aging-induced changes in cardiac hypertrophy and function. GPER deletion removed the sex difference in PWV as well as ex vivo stiffness in adult mice. The sex difference in blood pressure was absent in KO mice and was associated with endothelial dysfunction in females. These findings indicate that the impact of aging on arterial stiffening and endothelial function is not the same in male and female mice. Moreover, nongenomic estrogen signaling through GPER impacted vascular phenotype differently in male and female mice. Delineating sex differences in vascular changes during healthy aging is an important first step in improving early detection and sex-specific treatments in our aging population.

Aging is a nonmodifiable risk factor for cardiovascular disease associated with arterial stiffening and cardiac dysfunction. We previously showed an important role for G protein‐coupled estrogen receptor (GPER) in modulating female protection from arterial stiffening. In this study, we hypothesized that aging would decrease vascular compliance in GPER knockout (ko) mice to a greater extent than wildtype (wt) mice. Adult (25 weeks) and middle‐aged (57 weeks) mice were subjected to high frequency ultrasound for pulse wave velocity (PWV), and structural remodeling in the carotid artery was assessed with biaxial pressure myography followed by constitutive modeling. Data was analyzed by 3‐way ANOVA and Sidak’s post hoc tests. As we previously showed, PWV was significantly lower in adult female wt mice versus their male counterparts (1.4 ± 0.05 vs. 1.1 ± 0.02 m/s; P=0.0004) but was not different in adult ko mice (1.6 ± 0.07 vs 1.4 ± 0.08, P=0.20). Interestingly, PWV increased with aging in female but not male wt mice, so that the sex difference was lost (1.7 ± 0.03 vs 1.7 ± 0.06, P=0.99). Despite the lower PWV, circumferential linearized material stiffness in passive conditions was elevated in adult female wt versus male wt (1.9 ± 0.2 vs. 1.4 ± 0.2 MPa; P=0.02), but this sex difference was lost in middle‐aged wt mice (1.3 ± 0.1 vs. 1.2 ± 0.1 MPa; P=0.87). Aging significantly increased the ratio of thick to thin collagen only in male wt and female ko groups. We found that females but not males experience an increase in PWV at middle‐age along with a decrease in circumferential stiffness. In contrast, female GPER mice already display a higher PWV as adults, indicating that GPER deletion in females is a model of accelerated vascular aging.

Hypertensive patients frequently show a lack of 10% decline in nighttime (or nondipping) blood pressure (BP), which is a strong predictor of a cardiovascular event. Clock genes are essential regulators in diurnal BP in the vasculature during hypertension. However, little is known if vascular circadian clock factors influence BP rhythms in a sex-dependent manner. To test the hypothesis that AngII-induced hypertension disrupts the expression pattern of peripheral clock genes, male and female C57Bl/6J mice were subjected to AngII (700 ng/kg/min) infusion for two weeks. BP was measured by radiotelemetry (N=7/group). After two weeks, the animals were sacrificed during daytime or nighttime, and aortas were assessed for expression of clock genes (Per1, Per2, Bmal1) and estrogen receptors (ERα and GPER). Two-way ANOVA was used to compare light-dark cycle and AngII treatment effects. Hypertensive females (MAP: day 121 ± 9 vs. night 126 ± 10 mmHg, P=0.39; 7.3% dipping) but not males (MAP: day 128 ± 3 vs. night 140 ± 2 mmHg, P=0.01; >10% dipping) developed a non-dipping phenotype. AngII-hypertension amplified the circadian pattern of Per1 in the male aorta but removed the Per1 circadian variation in the female aorta. AngII did not alter Per2 oscillation in the aorta of either sex. Bmal1 patterns were also amplified in males and reversed in females. In response to AngII, aortic ERα expression was augmented during the day in females (control 287 ± 14 vs. AngII 413 ± 42 copies/ng RNA; P=0.01) but not in males (P=0.34). Furthermore, GPER expression exhibited a circadian oscillation in control (day 9 ± 1 vs. night 20 ± 7.6 copies/RNA ng) and hypertensive males (day 12 ± 17 vs. night 20 ± 5 copies/RNA ng, P=0.04), but not in females. In conclusion, sex differences exist in non-dipping BP patterns and circadian disruption of estrogen receptors and clock genes Per1 and Bmal1 in the vasculature after AngII treatment. This data suggest that females may be more vulnerable to circadian disruption during hypertension.