Abstract-Heat shock protein 72 (HSP72) is a stress-inducible protein capable of protecting a variety of cells from toxins, thermal stress, and ischemic injury. The cytoprotective role and mechanism of action of HSP72 in renal cell ischemic injury remain unclear. To study this, HSP72 was introduced (liposomal transfer) or induced (thermal stress, 43°Cϫ1 hour) in renal tubular cells (LLC-PK1) with Western blot confirmation. Cells were subjected to simulated ischemia 24 hours after liposomal HSP72 transfer or thermal stress, and the effect of HSP72 on nuclear factor-B (NF-B) activation (electrophoretic mobility shift assay and immunohistochemistry), IB␣ production (Western blot), postischemic tumor necrosis factor-␣ (TNF-␣) production (RT-PCR), and apoptosis (TUNEL assay) were determined. In separate experiments, the role of TNF-␣ in apoptosis was determined (anti-TNF-␣ neutralizing antibody). Results demonstrated that both liposomal transfer of HSP72 and thermal induction of HSP72 prevented NF-B activation and translocation, TNF-␣ gene transcription, and subsequent ischemia-induced renal tubular cell apoptosis. Furthermore, TNF-␣ neutralization also inhibited ischemia-induced renal tubular cell apoptosis. These results indicate that liposomal delivery of HSP72 inhibits ischemia-induced renal tubular cell apoptosis by preventing NF-B activation and subsequent TNF-␣ production. Further elucidation of the mechanisms of HSP-induced cytoprotection may result in therapeutic strategies that limit or prevent ischemia-induced renal damage.

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Valvular and vascular calcification are common causes of cardiovascular morbidity and mortality. Developing effective treatments requires understanding the molecular underpinnings of these processes. Shear stress is thought to play a role in inhibiting calcification. Furthermore, NOTCH1 regulates vascular and valvular endothelium, and human mutations in NOTCH1 can cause calcific aortic valve disease. Here, we determined the genome-wide impact of altering shear stress and NOTCH signaling on aortic valve endothelium. mRNA-sequencing of human aortic valve endothelial cells (HAVECs) with or without knockdown of NOTCH1, in the presence or absence of shear stress, revealed NOTCH1-dependency of the atherosclerosis-related gene connexin 40 (GJA5), and numerous repressors of endochondral ossification. Among these, Matrix GLA Protein (MGP) is highly expressed in aortic valve and vasculature, and inhibits soft tissue calcification by sequestering bone morphogenetic proteins (BMPs). Altering NOTCH1 levels affected MGP mRNA and protein in HAVECs. Furthermore, shear stress activated NOTCH signaling and MGP in a NOTCH1-dependent manner. NOTCH1 positively regulated endothelial MGP in vivo through specific binding motifs upstream of MGP. Our studies suggest that shear stress activates NOTCH1 in primary human aortic valve endothelial cells leading to downregulation of osteoblast-like gene networks that play a role in tissue calcification.