Strategies for late phase preclinical and early clinical trials of senolytics.

Cellular senescence and the hallmarks of aging contribute to age-related disease and dysfunction. The Unitary Theory of Fundamental Aging Mechanisms highlights the interdependence among the hallmarks of aging and suggests that by intervening in one fundamental aging process, most or all of the other processes could be impacted. Accumulation of senescent cells is associated with frailty, cardiovascular disease, obesity, diabetes, cognitive decline, and other age- and/or chronic disease-related disorders, suggesting that senescent cells are a target for intervention. Early preclinical data using senolytics, agents that target senescent cells, show promising results in several aging and disease models. The first in-human trials using the senolytic combination of Dasatinib and Quercetin indicated reduced senescent cell burden in adipose tissue of diabetic kidney disease patients and improved physical function in patients with idiopathic pulmonary fibrosis. Clinical trials with other senolytics, including the flavonoid Fisetin and BCL-xL inhibitors, are underway. These results from preclinical and early clinical trials illustrate the potential of senolytics to alleviate age-related dysfunction and diseases. However, multiple clinical trials across different aging and disease models are desperately needed. Parallel trials across institutions through the Translational Geroscience Network are facilitating testing to determine whether senolytics can be translated into clinical application.

Impact of Senescent Cell Subtypes on Tissue Dysfunction and Repair: Importance and Research Questions.

Cellular senescence, first observed and defined through cell culture studies, is a cell fate associated with essentially permanent cell cycle arrest and that can be triggered by a variety of inducers. Emerging evidence suggests senescence is a dynamic process with diverse functional characteristics. Depending on the tissue, type of inducer, and time since induction, senescent cells can promote tissue repair and re-modeling, prevent tumor development, or contribute to age-related disorders and chronic diseases, including cancers. Senescent cell characteristics appear to depend on multiple factors and be influenced by the milieu and other senescent cells locally and at a distance. We review diverse phenotypes of senescent cells originating from different cell types, senescence inducers over time since induction of senescence, and across conditions and diseases. This background is essential to inform further understanding about senescent cell subtypes and will point towards rational senescence-modulating strategies for achieving therapeutic benefit.

Adventitial delivery of nanoparticles encapsulated with 1α, 25-dihydroxyvitamin D3 attenuates restenosis in a murine angioplasty model.

Percutaneous transluminal angioplasty (PTA) of stenotic arteriovenous fistulas (AVFs) is performed to maintain optimal function and patency. The one-year patency rate is 60% because of venous neointimal hyperplasia (VNH) and venous stenosis (VS) formation. Immediate early response gene X-1 (Iex-1) also known as Ier3 increases in response to wall shear stress (WSS), and can cause VNH/VS formation in murine AVF. In human stenotic samples from AVFs, we demonstrated increased gene expression of Ier3. We hypothesized that 1α, 25-dihydroxyvitamin D3, an inhibitor of IER3 delivered as 1α, 25-dihydroxyvitamin D3 encapsulated in poly lactic-co-glycolic acid (PLGA) nanoparticles loaded in Pluronic F127 hydrogel (1,25 NP) to the adventitia of the stenotic outflow vein after PTA would decrease VNH/VS formation by reducing Ier3 and chemokine (C-C motif) ligand 2 (Ccl2) expression. In our murine model of AVF stenosis treated with PTA, increased expression of Ier3 and Ccl2 was observed. Using this model, PTA was performed and 10-µL of 1,25 NP or control vehicle (PLGA in hydrogel) was administered by adventitial delivery. Animals were sacrificed at day 3 for unbiased whole genome transcriptomic analysis and at day 21 for immunohistochemical analysis. Doppler US was performed weekly after AVF creation. At day 3, significantly lower gene expression of Ier3 and Ccl2 was noted in 1,25 NP treated vessels. Twenty-one days after PTA, 1,25 NP treated vessels had increased lumen vessel area, with decreased neointima area/media area ratio and cell density compared to vehicle controls. There was a significant increase in apoptosis, with a reduction in CD68, F4/80, CD45, pro-inflammatory macrophages, fibroblasts, Picrosirius red, Masson's trichrome, collagen IV, and proliferation accompanied with higher wall shear stress (WSS) and average peak velocity. IER3 staining was localized to CD68 and FSP-1 (+) cells. After 1,25 NP delivery, there was a decrease in the proliferation of α-SMA (+) and CD68 (+) cells with increase in the apoptosis of FSP-1 (+) and CD68 (+) cells compared to vehicle controls. RNA sequencing revealed a decrease in inflammatory and apoptosis pathways following 1,25 NP delivery. These data suggest that adventitial delivery of 1,25 NP reduces VNH and venous stenosis formation after PTA.

Experimental murine arteriovenous fistula model to study restenosis after transluminal angioplasty.

Percutaneous transluminal angioplasty (PTA) is a very common interventional treatment for treating stenosis in arteriovenous fistula (AVF) used for hemodialysis vascular access. Restenosis occurs after PTA, resulting in vascular lumen loss and a decrease in blood flow. Experimental animal models have been developed to study the pathogenesis of stenosis, but there is no restenosis model after PTA of stenotic AVF in mice. Here, we describe the creation of a murine model of restenosis after angioplasty of a stenosis in an AVF. The murine restenosis model has several advantages, including the rapid development of restenotic lesions in the vessel after angioplasty and the potential to evaluate endovascular and perivascular therapeutics for treating restenosis. The protocol includes a detailed description of the partial nephrectomy procedure to induce chronic kidney disease, the AVF procedure for development of de novo stenosis and the angioplasty treatment associated with progression of restenosis. We monitored the angioplasty-treated vessel for vascular patency and hemodynamic changes for a period of 28 d using ultrasound. Vessels were collected at different time points and processed for histological analysis and immunostaining. This angioplasty model, which can be performed with basic microvascular surgery skills, could be used to identify potential endovascular and perivascular therapies to reduce restenosis after angioplasty procedures.

Differences in Transforming Growth Factor-ß1/BMP7 Signaling and Venous Fibrosis Contribute to Female Sex Differences in Arteriovenous Fistulas.

Background Women have decreased hemodialysis arteriovenous fistula (AVF) maturation and patency rates. We determined the mechanisms responsible for the sex-specific differences in AVF maturation and stenosis formation by performing whole transcriptome RNA sequencing with differential gene expression and pathway analysis, histopathological changes, and in vitro cell culture experiments from male and female smooth muscle cells. Methods and Results Mice with chronic kidney disease and AVF were used. Outflow veins were evaluated for gene expression, histomorphometric analysis, Doppler ultrasound, immunohistologic analysis, and fibrosis. Primary vascular smooth muscle cells were collected from female and male aorta vessels. In female AVFs, RNA sequencing with real-time polymerase chain reaction analysis demonstrated a significant decrease in the average gene expression of BMP7 (bone morphogenetic protein 7) and downstream IL17Rb (interleukin 17 receptor b), with increased transforming growth factor-ß1 (Tgf-ß1) and transforming growth factor-ß receptor 1 (Tgfß-r1). There was decreased peak velocity, negative vascular remodeling with higher venous fibrosis and an increase in synthetic vascular smooth muscle cell phenotype, decrease in proliferation, and increase in apoptosis in female outflow veins at day 28. In vitro primary vascular smooth muscle cell experiments performed under hypoxic conditions demonstrated, in female compared with male cells, that there was increased gene expression of Tgf-ß1, Tgfß-r1, andCol1 with increased migration. Conclusions In female AVFs, there is decreased gene expression of BMP7 and IL17Rb with increased Tgf-ß1 and Tgfß-r1, and the cellular and vascular differences result in venous fibrosis with negative vascular remodeling.

Therapeutic Effect of Adipose Derived Mesenchymal Stem Cell Transplantation in Reducing Restenosis in a Murine Angioplasty Model.

BACKGROUND: Percutaneous transluminal angioplasty (PTA) is the first line of treatment for stenosis in the arteriovenous fistula (AVF) created to provide access for hemodialysis, but resenosis still occurs. Transplants of adipose-derived mesenchymal stem cells (AMSCs) labeled with green fluorescent protein (GFP) to the adventitia could reduce pro-inflammatory gene expression, possibly restoring patency in a murine model of PTA for venous stenosis. METHODS: Partial nephrectomy of male C57BL/6J mice induced CKD. Placement of the AVF was 28 days later and, 14 days after that, PTA of the stenotic outflow vein was performed with delivery of either vehicle control or AMSCs (5×105) to the adventitia of the vein. Mice were euthanized 3 days later and gene expression for interleukin-1 beta (IL-1ß) and tumor necrosis factor-alpha TNF-α) analyzed, and histopathologic analysis performed on day 14 and 28. GFP (+) AMSCs were tracked after transplantation for up to 28 days and Doppler ultrasound performed weekly after AVF creation. RESULTS: Gene and protein expression of IL-1ß and TNF-α, fibrosis, proliferation, apoptosis and smooth muscle actin decreased, and the proportions of macrophage types (M2/M1) shifted in a manner consistent with less inflammation in AMSC-transplanted vessels compared to controls. After PTA, AMSC-treated vessels had significantly higher wall shear stress, average peak, and mean velocity, with increased lumen vessel area and decreased neointima/media area ratio compared to the control group. At 28 days after delivery, GFP (+) AMSC were present in the adventitia of the outflow vein. CONCLUSIONS: AMSC-treated vessels had improved vascular remodeling with decreased proinflammatory gene expression, inflammation, and fibrotic staining compared to untreated vessels.

Effect of sex differences in treatment response to angioplasty in a murine arteriovenous fistula model.

Failure to mature and venous neointimal hyperplasia formation are the two major causes of hemodialysis arteriovenous fistula (AVF) vascular access failure. Percutaneous transluminal angioplasty (PTA) is the firstline treatment for both of these conditions, but, clinically, women have decreased patency rates compared with men. The hypothesis to be tested in the present study was that female mice after PTA of venous areas of higher intimal thickening have increased gene expression of transforming growth factor-ß1 (TGF-ß1) and TGF-ß receptor 1 (TGFß-R1) accompanied with histological changes of fibrosis compared with male mice. Seventeen male and eighteen female C57BL/6J mice were used in this study. Chronic kidney disease was induced by partial nephrectomy, and, 28 days later, an AVF was created to connect the left carotid artery to the right jugular vein. Two weeks later, the higher intimal thickening area was treated with PTA, and mice were euthanized 3 days later for gene expression analysis or 14 days later for histopathological analysis. Doppler ultrasound was performed weekly after AVF creation. At day 3, female AVF had significantly higher average gene expression of TGF-ß1 and TGFß-R1 compared with male AVF. At day 14, female outflow veins had a smaller venous diameter, lumen vessel area, decreased wall shear stress, lower average peak systolic velocity, and an increased neointima area-to-media area ratio. Moreover, female outflow veins showed a significant increase in α-smooth muscle actin and fibroblast-specific protein-1. There was a decrease in M1/M2 with an increase in CD68.