Tubular overexpression of Angiopoietin-1 attenuates renal fibrosis

Emerging evidence has highlighted the pivotal role of microvasculature injury in the development and progression of renal fibrosis. Angiopoietin-1 (Ang-1) is a secreted vascular growth factor that binds to the endothelial-specific Tie2 receptor. Ang-1/Tie2 signaling is critical for regulating blood vessel development and modulating vascular response after injury, but is dispensable in mature, quiescent vessels. Although dysregulation of vascular endothelial growth factor (VEGF) signaling has been well studied in renal pathologies, much less is known about the role of the Ang-1/Tie2 pathway in renal interstitial fibrosis. Previous studies have shown contradicting effects of overexpressing Ang-1 systemically on renal tubulointerstitial fibrosis when different engineered forms of Ang-1 are used. Here, we investigated the impact of site-directed expression of native Ang-1 on the renal fibrogenic process and peritubular capillary network by exploiting a conditional transgenic mouse system [Pax8-rtTA/(TetO)7 Ang-1] that allows increased tubular Ang-1 production in adult mice. Using a murine unilateral ureteral obstruction (UUO) fibrosis model, we demonstrate that targeted Ang-1 overexpression attenuates myofibroblast activation and interstitial collagen I accumulation, inhibits the upregulation of transforming growth factor β1 and subsequent phosphorylation of Smad 2/3, dampens renal inflammation, and stimulates the growth of peritubular capillaries in the obstructed kidney. Our results suggest that Ang-1 is a potential therapeutic agent for targeting microvasculature injury in renal fibrosis without compromising the physiologically normal vasculature in humans

Genetics, Treatment, and New Technologies of Hormone Receptor-Positive Breast Cancer

The current molecular classification divides breast cancer into four major subtypes, including luminal A, luminal B, HER2-positive, and basal-like, based on receptor gene expression profiling. Luminal A and luminal B are hormone receptor (HR, estrogen, and/or progesterone receptor)-positive and are the most common subtypes, accounting for around 50–60% and 15–20% of the total breast cancer cases, respectively. The drug treatment for HR-positive breast cancer includes endocrine therapy, HER2-targeted therapy (depending on the HER2 status), and chemotherapy (depending on the risk of recurrence). In this review, in addition to classification, we focused on discussing the important aspects of HR-positive breast cancer, including HR structure and signaling, genetics, including epigenetics and gene mutations, gene expression-based assays, the traditional and new drugs for treatment, and novel or new uses of technology in diagnosis and treatment. Particularly, we have summarized the commonly mutated genes and abnormally methylated genes in HR-positive breast cancer and compared four common gene expression-based assays that are used in breast cancer as prognostic and/or predictive tools in detail, including their clinical use, the factors being evaluated, patient demographics, and the scoring systems. All these topic discussions have not been fully described and summarized within other research or review articles

Comparison of on-campus and virtual self-assessment outcomes for incoming Appalachian STEM undergraduates’ first research experience

The First2 Network is an alliance of higher education institutions across the State of West Virginia striving to improve science, technology, engineering or math (STEM) education by supporting rural, first-generation, and underrepresented college students pursuing STEM majors.  Over the summers of 2019 and 2020, the First2 Network delivered two-week summer research immersion experiences at various institutions throughout West Virginia. The 2019 program was delivered on-campus at four universities while the 2020 program was delivered virtually, due to the COVID-19 pandemic, across nine sites. Before and after the immersion experience, students who participated in the program completed a variety of survey questionnaires for the assessment of their interests, expectations, identity and belonging in STEM. We found that the in-person experience in 2019 had better outcomes compared to the virtual experience, suggesting students conducting research directly under their faculty supervisors in-person and on-site will have a more positive impact on their STEM education and career. However, participation in the virtual format still resulted in an improvement in belonging and STEM identity, indicating that connecting with students remotely is still worthwhile when it is the most viable option. The student population in Appalachia faces a number of academic barriers, so there is much to gain by finding new ways to reach as many students as possible with early career development programs

Knockdown of HSPA9 induces TP53-dependent apoptosis in human hematopoietic progenitor cells.

Myelodysplastic syndromes (MDS) are the most common adult myeloid blood cancers in the US. Patients have increased apoptosis in their bone marrow cells leading to low peripheral blood counts. The full complement of gene mutations that contribute to increased apoptosis in MDS remains unknown. Up to 25% of MDS patients harbor and acquired interstitial deletion on the long arm of chromosome 5 [del(5q)], creating haploinsufficiency for a large set of genes including HSPA9. Knockdown of HSPA9 in primary human CD34+ hematopoietic progenitor cells significantly inhibits growth and increases apoptosis. We show here that HSPA9 knockdown is associated with increased TP53 expression and activity, resulting in increased expression of target genes BAX and p21. HSPA9 protein interacts with TP53 in CD34+ cells and knockdown of HSPA9 increases nuclear TP53 levels, providing a possible mechanism for regulation of TP53 by HSPA9 haploinsufficiency in hematopoietic cells. Concurrent knockdown of TP53 and HSPA9 rescued the increased apoptosis observed in CD34+ cells following knockdown of HSPA9. Reduction of HSPA9 below 50% results in severe inhibition of cell growth, suggesting that del(5q) cells may be preferentially sensitive to further reductions of HSPA9 below 50%, thus providing a genetic vulnerability to del(5q) cells. Treatment of bone marrow cells with MKT-077, an HSPA9 inhibitor, induced apoptosis in a higher percentage of cells from MDS patients with del(5q) compared to non-del(5q) MDS patients and normal donor cells. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to TP53 activation and increased apoptosis observed in del(5q)-associated MDS

HSPA9 inhibitor MKT-077 reduces cell growth, induces apoptosis, and increases p21 and BAX expression in CD34+ cells.

<p>(A) Immunoblot of HSPA9, p21, BAX and TP53 following MKT-077 treatment of CD34+ cells grown in erythroid culture conditions for 5 days. (B) CD34+ cells were seeded at equal concentrations in erythroid culture conditions, and the total number of cells was counted daily in the presence of MKT-077. The fold change in cell counts was calculated relative to the number of cells on day 1 (n = 3) (0.5 μM versus control, p<0.001; 2 μM versus control, p<0.001). (C) Quantification of Annexin V+ cells (a surrogate for apoptosis) following treatment of CD34+ cells with various concentrations of MKT-077 for 5 days (n = 3). ***p<0.001.</p

Knockdown of HSPA9 increases TP53 expression and activity in CD34+ cells.

<p>(A) Representative immunoblot of HSPA9 and TP53 protein in CD34+ cells transduced with shRNAs targeting a control gene (GFP) or HSPA9 after 7 days in erythroid culture conditions. (B) Transduced cells were seeded at equal concentrations in erythroid culture media and the total number of cells was counted daily in the presence of puromycin selection. The fold change in cell counts was calculated relative to the number of cells on day 1 (n = 3 independent replicates for each shRNA). (C) Quantification of Annexin V+ cells (a surrogate for apoptosis) following knockdown of HSPA9 in CD34+ cells (n = 3 for each shRNA). (D) Gene Set Enrichment Analysis (GSEA) enrichment score plot for 26 annotated TP53-induced genes. Individual genes in the gene set are represented by a black vertical bar in the middle of the plot. (E) TP53 luciferase reporter assay showed a dose-dependent increase in TP53 activity following HSPA9 knockdown (n = 3). LUC, luciferase. * p<0.05, ** p<0.01, *** p<0.001.</p

HSPA9 and TP53 interact in CD34+ cells and HSPA9 knockdown increases nuclear TP53 levels.

<p>(A) Representative immunoblots of TP53 and HSPA9 following immunoprecipitation with anti-TP53 or an IgG control. (B) Representative immunoblots of TP53 from the cytoplasmic and nuclear fractions of cell lysates following knockdown of HSPA9 in CD34+ cells grown in erythroid culture conditions for 5 days.</p

Reduction of TP53 inhibits apoptosis induced by HSPA9 knockdown.

<p>(A) Immunoblot of TP53 following knockdown of TP53 by shRNAs in CD34+ cells grown in erythroid culture conditions for 4 days. (B) CD34+ cells grown in erythroid culture conditions were co-transduced with lentiviral constructs carrying an shRNA targeting TP53 with a hygromycin resistance gene (e.g., shGFP, shTP53-3, or shTP53-4) and an shRNA targeting HSPA9 with a puromycin resistance gene (shGFP, sh433, or sh960). Cells were grown in the presence of both hygromycin and puromycin and the fold change in the percentage of Annexin V+ cells was measured by flow cytometry and normalized to the shGFP-hygromycin/shGFP-puromycin transduced cells (n = 3 technical replicates, representative of 2 independent experiments). *p<0.05, **p<0.01,***p<0.001.</p

Knockdown of HSPA9 increases expression of the TP53 target genes p21 and BAX in CD34+ cells.

<p>(A) Representative immunoblot of p21 and BAX following knockdown of HSPA9 in CD34+ cells. (B) Gene Set Enrichment Analysis (GSEA) enrichment score plot for 13 annotated p21-inhibited genes. Individual genes in the gene set are represented by a black vertical bar in the middle of the plot.</p