Angiogenesis is a complex process coordinating cell migration, proliferation, and lumen formation. Changes to the microenvironment regulate angiogenesis through mechanotransduction and cytokine signals. In pulmonary hypertension, something in the process becomes abnormal, resulting in changes to the microenvironment and the formation of a glomerulus of dysfunctional capillaries, called a plexiform lesion. Endothelial cells, expressing CD117 (CD117+ EC clones) increase in the plexiform lesions of pulmonary hypertension, independent of pro-angiogenic VEGF signaling. We hypothesize that the mechanical environment and the macromolecular composition of the extracellular matrix, both, contribute to the aberrant angiogenesis. When we changed the mechanical environment, we changed the angiogenic potential and cellular phenotype of CD117+ Endothelial cell clones. Turbulent flow, pathologic substrate stiffness, and pathologic stretch increased Endothelial-to-mesenchymal markers, such as acta2, cnn1, snail, and slug in CD117+ EC clones while CD117- ECs showed minimal change. We perturbed the mechanical environment of CD117+ EC clones and identified changes in Bone Morphogenic Protein-2, an often overlooked pro-angiogenic cytokine. We coupled changes in the mechanical environment to Rho GTPase intracellular signaling, to predict how changes to the mechanotransduction would affect angiogenesis through a computational model. In our model of angiogenesis, we found vessel synchronicity to depend on both which cell undergoes mitosis, and also at which phase of GTPase cycling the cell undergoes mitosis. We believe changes to the GTPase cycling may be the mechanism linking mechanotransduction to the abnormal vessels found in pulmonary hypertension. We are the first group to look at the role of the ECM composition, independent of stiffness. Our results show diseased ECM composition alone leads to phenotypic changes indicative of PH progression. In conclusion, these results provide a possible cytokine implicated in the mechanotransduction of PH, established a computational model of angiogenesis which provides a mechanotransduction mechanism of disease progression, and established that the ECM composition alone is capable of phenotypic changes leading to disease progression.

Pigmentation plays a protective role against damage caused by ultraviolet (UV) irradiation. Humans with fair skin and light hair have a higher susceptibility to UV-induced DNA damage that can lead to the development of skin cancers. The melanocytes found in the skin and hair follicles depend on different signaling molecules for their proper development and pigment production. α-Melanocyte Stimulating Hormone (α-msh) binds to the Melanocortin 1 receptor (Mc1r) to regulate pigment production and the switch between eumelanin and pheomelanin. Lethal yellow mice (Ay) overexpress the agouti signaling protein, which inhibits the binding of α-msh, resulting in a yellow coat color phenotype. Endothelin 3 (Edn3) encodes for a ligand involved in melanocyte development by regulating the differentiation, proliferation and migration of melanocyte precursors. A tetracycline inducible transgenic mouse in which Edn3 was placed under the keratin 5 promoter (K5-tTA;TRE-Edn3-lacZ) displays a hyperpigmentation phenotype due to the accumulation of melanocytes in the skin and an increase in hair pigment. Comparative analysis of dorsal hairs from Ay and Ay; K5-tTA;TRE-Edn3-lacZ mice using high performance liquid chromatography showed that transgenic Edn3 expression significantly increased both eumelanin and pheomelanin. No significant difference in the number of follicular melanocytes between Edn3 transgenic and non-transgenic mice was evidenced by immunofluorescence using an antibody against Tyrosinase related protein 1. Gene expression analysis of hair follicles showed that Edn3 upregulates the expression of melanogenic genes. Deactivation of transgenic Edn3 is possible with doxycycline (dox) treatment. To test if transgenic Edn3 expression is required to rescue and maintain a dark pigmentation phenotype in Ay mice, dox was administered during embryonic and postnatal development to manipulate transgenic Edn3 expression. Results showed that transgenic Edn3 expression is required to maintain a dark pigmentation phenotype after birth but is independent of a developmental requirement. Transgenic Edn3 expression in Mc1re/e mice also resulted in a darkened coat color. Our results indicate that the paracrine expression of Edn3 from keratinocytes is capable of generating and maintaining a dark coat color by the regulation of melanogenic genes independent of Mc1r signaling. The results of this study may open new approaches to the treatment of hypopigmentation disorders.

Overweight and obesity are associated with Type 2 Diabetes, non-alcoholic fatty liver disease, cardiovascular disease and cancer, but all fat is not equal as storing excess lipid in subcutaneous white adipose tissue (SWAT) is more metabolically favorable than in visceral fat. Here, we uncover a critical role for mTORC2 in setting SWAT lipid handling capacity. We find that subcutaneous white preadipocytes differentiating without the essential mTORC2 subunit Rictorexpress mature adipocyte markers but develop a striking lipid storage defect. In vivo,this results in smaller adipocytes, reduced tissue size, lipid re-distribution to visceral and brown fat, and sex-distinct effects on systemic metabolic fitness. Mechanistically, mTORC2 promotes transcriptional upregulation of select lipid metabolism genes controlled by PPARgand ChREBP. These include genes that control lipid uptake, synthesis, and degradation pathways as well as Akt2, the gene encoding its substrate and insulin effector. Finally, we reveal a potential novel mTORC2 target, ACSS2, which might control intracellular acetyl-CoA availability and regulate metabolic gene expression by altering histone modification in white adipocytes. Exploring this pathway may uncover strategies to promote safe lipid storage and improve insulin sensitivity.