In calcific aortic valve disease (CAVD), microcalcifications originating from nanoscale calcifying vesicles disrupt the aortic valve (AV) leaflets, which consist of three (biomechanically) distinct layers: the fibrosa, spongiosa, and ventricularis. CAVD has no pharmacotherapy and lacks in vitro models as a result of complex valvular biomechanical features surrounding resident mechanosensitive valvular interstitial cells (VICs). We measured layer-specific mechanical properties of the human AV and engineered a three-dimensional (3D)-bioprinted CAVD model that recapitulates leaflet layer biomechanics for the first time. Human AV leaflet layers were separated by microdissection, and nanoindentation determined layer-specific Young’s moduli. Methacrylated gelatin (GelMA)/methacrylated hyaluronic acid (HAMA) hydrogels were tuned to duplicate layer-specific mechanical characteristics, followed by 3D-printing with encapsulated human VICs. Hydrogels were exposed to osteogenic media (OM) to induce microcalcification, and VIC pathogenesis was assessed by near infrared or immunofluorescence microscopy. Median Young’s moduli of the AV layers were 37.1, 15.4, and 26.9 kPa (fibrosa/spongiosa/ventricularis, respectively). The fibrosa and spongiosa Young’s moduli matched the 3D 5% GelMa/1% HAMA UV-crosslinked hydrogels. OM stimulation of VIC-laden bioprinted hydrogels induced microcalcification without apoptosis. We report the first layer-specific measurements of human AV moduli and a novel 3D-bioprinted CAVD model that potentiates microcalcification by mimicking the native AV mechanical environment. This work sheds light on valvular mechanobiology and could facilitate high-throughput drug-screening in CAVD.

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The appearance of constitutively active androgen receptor splice variants (AR-Vs) has been proposed as one of the causes of castration-resistant prostate cancer (CRPC). However, the underlying mechanism of AR-Vs in CRPC transcriptional regulation has not been defined. A distinct transcriptome enriched with cell cycle genes, e.g. UBE2C, has been associated with AR-Vs, which indicates the possibility of an altered transcriptional mechanism when compared to full-length wild-type AR (ARfl). Importantly, a recent study reported the critical role of p-MED1 in enhancing UBE2C expression through a locus looping pattern, which only occurs in CRPC but not in androgen-dependent prostate cancer (ADPC). To investigate the potential correlation between AR-V and MED1, in the present study we performed protein co-immunoprecipitation, chromatin immunoprecipitation, and cell proliferation assays and found that MED1 is necessary for ARv567es induced UBE2C up-regulation and subsequent prostate cancer cell growth. Furthermore, p-MED1 is bound to ARv567es independent of full-length AR; p-MED1 has higher recruitment to UBE2C promoter and enhancer regions in the presence of ARv567es. Our data indicate that p-MED1 serves as a key mediator in ARv567es induced gene expression and suggests a mechanism by which AR-Vs promote the development and progression of CRPC.

Endoplasmic reticulum (ER) stress response is an adaptive program to cope with cellular stress that disturbs the function and homeostasis of ER, which commonly occurs during cancer progression to late stage. Late-stage cancers, mostly requiring chemotherapy, often develop treatment resistance. Chemoresistance has been linked to ER stress response; however, most of the evidence has come from studies that correlate the expression of stress markers with poor prognosis or demonstrate proapoptosis by the knockdown of stress-responsive genes. Since ER stress in cancers usually persists and is essentially not induced by genetic manipulations, we used low doses of ER stress inducers at levels that allowed cell adaptation to occur in order to investigate the effect of stress response on chemoresistance. We found that prolonged tolerable ER stress promotes mesenchymal–epithelial transition, slows cell-cycle progression, and delays the S-phase exit. Consequently, cisplatin-induced apoptosis was significantly decreased in stress-adapted cells, implying their acquisition of cisplatin resistance. Molecularly, we found that proliferating cell nuclear antigen (PCNA) ubiquitination and the expression of polymerase η, the main polymerase responsible for translesion synthesis across cisplatin-DNA damage, were up-regulated in ER stress-adaptive cells, and their enhanced cisplatin resistance was abrogated by the knockout of polymerase η. We also found that a fraction of p53 in stress-adapted cells was translocated to the nucleus, and that these cells exhibited a significant decline in the level of cisplatin-DNA damage. Consistently, we showed that the nuclear p53 coincided with strong positivity of glucose-related protein 78 (GRP78) on immunostaining of clinical biopsies, and the cisplatin-based chemotherapy was less effective for patients with high levels of ER stress. Taken together, this study uncovers that adaptation to ER stress enhances DNA repair and damage tolerance, with which stressed cells gain resistance to chemotherapeutics.