Browsing by Subject "endothelium"

Hutchison-Gilford Progeria Syndrome (HGPS) is a rare, accelerated aging disorder caused by nuclear accumulation of progerin, an altered form of the Lamin A gene. The primary causes of death are stroke and cardiovascular disease at an average age of 14 years. It is known that loss or malfunction of smooth muscle cells (SMCs) in the vasculature leads to cardiovascular defects, however, the exact mechanisms are still not understood. The contribution of other vascular cell types, such as endothelial cells, is still not known due to the current limitations of studying such a rare disorder. Due to limitations of 2D cell culture, mouse models, and the limited HGPS patient pool, there is a need to develop improved models of HGPS to better understand the development of the disease and discover novel therapeutics.

To address these limitations, we produced a functional, three-dimensional tissue model of HGPS that replicates an arteriole-scale tissue engineered blood vessel (TEBV) using induced pluripotent stem cell (iPSC)-derived cell sources from HGPS patients. To isolate the specific effects of HGPS SMCs, we initially used human cord blood-derived endothelial progenitor cells (hCB-EPCs) from a separate, healthy donor and iPSC-derived SMCs (iSMCs). TEBVs fabricated from HGPS patient iSMCs and hCB-EPCs (HGPS iSMC TEBVs) showed disease attributes such as reduced vasoactivity, increased medial wall thickness, increased calcification, excessive extracellular matrix protein deposition, and cell apoptosis relative to TEBVs fabricated from primary mesenchymal stem cells (MSCs) and hCB-EPCs or normal patient iSMCs with hCB-EPCs. Treatment of HGPS iSMC TEBVs for one week with the rapamycin analog Everolimus (RAD001), increased HGPS iSMC TEBV vasoactivity and iSMC differentiation in TEBVs.

To improve the sensitivity of our HGPS TEBV model and study the effects of endothelial cells on the HGPS cardiovascular phenotype, we adopted a modified differentiation protocol to produce iPSC-derived vascular smooth muscle cells (viSMCs) and endothelial cells (viECs) from normal and Progeria patient iPSC lines to create iPSC-derived vascular TEBVs (viTEBVs). Normal viSMCs and viECs showed structural and functional characteristics of vascular SMCs and ECs in 2D culture, while HGPS viSMCs and viECs showed various disease characteristics and reduced function compared to healthy controls. Normal viTEBVs had comparable structure and vasoactivity to MSC TEBVs, while HGPS viTEBVs showed reduced vasoactivity, increased vessel wall thickness, calcification, apoptosis and excess ECM deposition. In addition, HGPS viTEBVs showed markers of cardiovascular disease associated with the endothelium such as decreased response to acetylcholine, increased inflammation, and altered expression of flow-associated genes.

The treatment of viTEBVs with multiple Progeria therapeutics was evaluated to determine the potential of the HGPS viTEBV model to serve as a platform for drug efficacy and toxicity testing as well as to further elucidate the mechanisms behind each drugs mode of action. Treatment of viTEBVs with therapeutic levels of the farnesyl-transferase inhibitor (FTI), Lonafarnib, or Everolimus improved different aspects of HGPS viTEBV structure and function. Treatment with Everolimus alone increased response to phenylephrine, improved SMC differentiation and cleared progerin through autophagy. Lonafarnib improved acetylcholine response, decreased ECM deposition, decreased calcification and improved nitric oxide production. Most significantly, combined therapeutic treatment with both drugs showed an additive effect by improving overall vasoactivity, increasing cell density, increasing viSMC and viEC differentiation, and decreasing calcification and apoptosis in treated HGPS viTEBVs. On the other hand, toxic doses of both drugs combined resulted in significantly diminished HGPS viTEBV function through increased cell death. In summary, this work shows the ability of a tissue engineered vascular model to serve as an in vitro personalized medicine platform to study HGPS and potentially other rare diseases of the vasculature using iPSC-derived cell sources. It has also further identified a potential role of the endothelium in HGPS. Finally, this HGPS viTEBV model has proven effective as a drug testing platform to determine therapeutic and toxic doses of proposed therapeutics based on their specific therapeutic effects on HGPS viTEBV structure and function.

Covering the metal surface of blood-contacting cardiovascular implants (stents, ventricular assist devices) with functional endothelium may reduce the incidence of clotting and restenosis complications and also reduce the need for risky anticoagulation therapy following implantation of such devices. We developed a novel cell therapy for seeding autologous endothelium onto blood-contacting vascular stents at the point of care to reduce thrombosis and stent restenosis. The proposed research tested the following hypotheses: (1) autologous endothelial cells (ECs) can spread on titanium (Ti) tubes and reduce thrombosis on the Ti surface in vivo; (2) shear stresses on the surfaces of an implanted carotid artery stent will be conducive to EC retention and function under arterial flow; and (3) nitinol stents seeded with ECs at the point of care will remain adherent and functional after stent deployment and arterial fluid shear stress conditions in vitro and in vivo. Based on the experiments reported herein, the primary conclusions of the dissertation are as follows: (1) autologous ECs significantly reduce thrombosis on Ti surfaces implanted into the bloodstream in vivo; (2) shear stresses on stent surfaces under carotid artery flow conditions are sufficiently low to be compatible with EC retention and function; (3) ECs seeded onto nitinol stents by infusion at the point of care are retained and spread to form a functional layer following deployment and arterial flow conditions both in vitro and in vivo.