RESUMEN
In this study, we present a versatile, scaffold-free approach to create ring-shaped engineered vascular tissue segments using human mesenchymal stem cell-derived smooth muscle cells (hMSC-SMCs) and endothelial cells (ECs). We hypothesized that incorporation of ECs would increase hMSC-SMC differentiation without compromising tissue ring strength or fusion to form tissue tubes. Undifferentiated hMSCs and ECs were co-seeded into custom ring-shaped agarose wells using four different concentrations of ECs: 0, 10, 20, and 30%. Co-seeded EC and hMSC rings were cultured in SMC differentiation medium for a total of 22 days. Tissue rings were then harvested for histology, western blotting, wire myography, and uniaxial tensile testing to examine their structural and functional properties. Differentiated hMSC tissue rings comprised of 20 and 30% ECs exhibited significantly greater SMC contractile protein expression, endothelin-1 (ET-1)-meditated contraction, and force at failure compared to the 0% EC rings. On average, the 0, 10, 20, and 30% EC rings exhibited a contractile force of 0.745 ± 0.117, 0.830 ± 0.358, 1.31 ± 0.353, and 1.67 ± 0.351 mN (mean ± SD) in response to ET-1, respectively. Additionally, the mean maximum force at failure for the 0, 10, 20, and 30% EC rings was 88.5 ± 36.2, 121 ± 59.1, 147 ± 43.1, and 206 ± 20.8 mN (mean ± SD), respectively. Based on these results, 30% EC rings were fused together to form tissue engineered blood vessels (TEBVs) and compared to 0% EC TEBV controls. The addition of 30% ECs in TEBVs did not affect ring fusion but did result in significantly greater SMC protein expression (calponin and smoothelin). In summary, co-seeding hMSCs with ECs to form tissue rings resulted in greater contraction, strength, and hMSC-SMC differentiation compared to hMSCs alone and indicates a method to create functional 3D human vascular cell co-culture model.
RESUMEN
BACKGROUND: RAB27A is a member of the RAS oncogene superfamily of GTPases and regulates cell secretory function. It, is expressed within blood vessels and perivascular adipose tissue. We hypothesized that loss of RAB27A would alter cardiovascular function. METHODS: Body weight of Rab27aash mice was measured from 2 to 18 months of age, along with glucose resorption at 6 and 12 months of age and glucose sensitivity at 18 months of age. Body weight and cellular and molecular features of perivascular adipose tissue and aortic tissue were examined in a novel C57BL/6J Rab27a null strain. Analyses included morphometric quantification and proteomic analyses. Wire myography measured vasoreactivity, and echocardiography measured cardiac function. Comparisons across ages and genotypes were evaluated via 2-way ANOVA with multiple comparison testing. Significance for myography was determined via 4-parameter nonlinear regression testing. RESULTS: Genome-wide association data linked rare human RAB27A variants with body mass index and glucose handling. Changes in glucose tolerance were observed in Rab27aash male mice at 18 months of age. In WT (wild-type) and Rab27a null male mice, body weight, adipocyte lipid area, and aortic area increased with age. In female mice, only body weight increased with age, independent of RAB27A presence. Protein signatures from male Rab27a null mice suggested greater associations with cardiovascular and metabolic phenotypes compared with female tissues. Wire myography results showed Rab27a null males exhibited increased vasoconstriction and reduced vasodilation at 8 weeks of age. Rab27a null females exhibited increased vasoconstriction and vasodilation at 20 weeks of age. Consistent with these vascular changes, male Rab27a null mice experienced age-related cardiomyopathy, with severe differences observed by 21 weeks of age. CONCLUSIONS: Global RAB27A loss impacted perivascular adipose tissue and thoracic aorta proteomic signatures, altered vasocontractile responses, and decreased left ventricular ejection fraction in mice.
Asunto(s)
Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas rab27 de Unión a GTP , Animales , Proteínas rab27 de Unión a GTP/genética , Proteínas rab27 de Unión a GTP/metabolismo , Masculino , Femenino , Ratones , Fenotipo , Tejido Adiposo/metabolismo , Vasodilatación , Vasoconstricción , Factores de Edad , Proteómica , Factores Sexuales , Aorta/metabolismo , Aorta/fisiopatología , HumanosRESUMEN
Perivascular adipose tissue (PVAT) regulates vascular function by secreting vasoactive substances. In mice, Notch signaling is activated in the PVAT during diet-induced obesity, and leads to the loss of the thermogenic phenotype and adipocyte whitening due to increased lipid accumulation. We used the Adiponectin-Cre (Adipoq-Cre) strain to activate a ligand-independent Notch1 intracellular domain transgene (N1ICD) to drive constitutive Notch signaling in the adipose tissues (N1ICD;Adipoq-Cre). We previously found that constitutive activation of Notch1 signaling in the PVAT phenocopied the effects of diet-induced obesity. To understand the downstream pathways activated by Notch signaling, we performed a proteomic analysis of the PVAT from control versus N1ICD;Adipoq-Cre mice. This comparison identified prominent changes in the protein signatures related to metabolism, adipocyte homeostasis, mitochondrial function, and ferroptosis. PVAT-derived stromal vascular fraction cells were derived from our mouse strains to study the cellular and molecular phenotypes during adipogenic induction. We found that cells with activated Notch signaling displayed decreased mitochondrial respiration despite similar levels of adipogenesis and mitochondrial number. We observed variable regulation of the proteins related to mitochondrial dynamics and ferroptosis, including PHB3, PINK1, pDRP1, and the phospholipid hydroperoxidase GPX4. Mitochondria regulate some forms of ferroptosis, which is a regulated process of cell death driven by lipid peroxidation. Accordingly, we found that Notch activation promoted lipid peroxidation and ferroptosis in PVAT-derived adipocytes. Because the PVAT phenotype is a regulator of vascular reactivity, we tested the effect of Notch activation in PVAT on vasoreactivity using wire myography. The aortae from the N1ICD;Adipoq-Cre mice had increased vasocontraction and decreased vasorelaxation in a PVAT-dependent and age-dependent manner. Our data provide support for the novel concept that increased Notch signaling in the adipose tissue leads to PVAT whitening, impaired mitochondrial function, increased ferroptosis, and loss of a protective vasodilatory signal. Our study advances our understanding of how Notch signaling in adipocytes affects mitochondrial dynamics, which impacts vascular physiology.