A cell atlas of thoracic aortic perivascular adipose tissue: a focus on mechanotransducers.

Perivascular adipose tissue (PVAT) is increasingly recognized for its function in mechanotransduction. However, major gaps remain in our understanding of the cells present in PVAT, as well as how different cells contribute to mechanotransduction. We hypothesized that snRNA-seq would reveal the expression of mechanotransducers, and test one (PIEZO1) to illustrate the expression and functional agreement between single-nuclei RNA sequencing (snRNA-seq) and physiological measurements. To contrast two brown tissues, subscapular brown adipose tissue (BAT) was also examined. We used snRNA-seq of the thoracic aorta PVAT (taPVAT) and BAT from male Dahl salt-sensitive (Dahl SS) rats to investigate cell-specific expression mechanotransducers. Localization and function of the mechanostransducer PIEZO1 were further examined using immunohistochemistry (IHC) and RNAscope, as well as pharmacological antagonism. Approximately 30,000 nuclei from taPVAT and BAT each were characterized by snRNA-seq, identifying eight major cell types expected and one unexpected (nuclei with oligodendrocyte marker genes). Cell-specific differential gene expression analysis between taPVAT and BAT identified up to 511 genes (adipocytes) with many (≥20%) being unique to individual cell types. Piezo1 was the most highly, widely expressed mechanotransducer. The presence of PIEZO1 in the PVAT but not the adventitia was confirmed by RNAscope and IHC in male and female rats. Importantly, antagonism of PIEZO1 by GsMTX4 impaired the PVAT's ability to hold tension. Collectively, the cell compositions of taPVAT and BAT are highly similar, and PIEZO1 is likely a mechanotransducer in taPVAT.NEW & NOTEWORTHY This study describes the atlas of cells in the thoracic aorta perivascular adipose tissue (taPVAT) of the Dahl-SS rat, an important hypertension model. We show that mechanotransducers are widely expressed in these cells. Moreover, PIEZO1 expression is shown to be restricted to the taPVAT and is functionally implicated in stress relaxation. These data will serve as the foundation for future studies investigating the role of taPVAT in this model of hypertensive disease.

Exploring structural effects in a new class of NRF2 inhibitors.

NRF2 is a transcription factor that controls the cellular response to various stressors, such as reactive oxygen and nitrogen species. As such, it plays a key role in the suppression of carcinogenesis, but constitutive NRF2 expression in cancer cells leads to resistance to chemotherapeutics and promotes metastasis. As a result, inhibition of the NRF2 pathway is a target for new drugs, especially for use in conjunction with established chemotherapeutic agents like carboplatin and 5-fluorouracil. A new class of NRF2 inhibitors has been discovered with substituted nicotinonitriles, such as MSU38225. In this work, the effects on NRF2 inhibition with structural changes were explored. Through these studies, we identified a few compounds with as good or better activity than the initial hit but with greatly improved solubility. The syntheses involved a variety of metal-catalyzed reactions, including titanium multicomponent coupling reactions and various Pd and Cu coupling reactions. In addition to inhibiting NRF2 activity, these new compounds inhibited the proliferation and migration of lung cancer cells in which the NRF2 pathway is constitutively activated.

A Novel Nrf2 Pathway Inhibitor Sensitizes Keap1-Mutant Lung Cancer Cells to Chemotherapy.

The nuclear factor erythroid-2-related factor 2 (Nrf2)-Keap1-ARE pathway, a master regulator of oxidative stress, has emerged as a promising target for cancer therapy. Mutations in NFE2L2, KEAP1, and related genes have been found in many human cancers, especially lung cancer. These mutations lead to constitutive activation of the Nrf2 pathway, which promotes proliferation of cancer cells and their resistance to chemotherapies. Small molecules that inhibit the Nrf2 pathway are needed to arrest tumor growth and overcome chemoresistance in Nrf2-addicted cancers. Here, we identified a novel small molecule, MSU38225, which can suppress Nrf2 pathway activity. MSU38225 downregulates Nrf2 transcriptional activity and decreases the expression of Nrf2 downstream targets, including NQO1, GCLC, GCLM, AKR1C2, and UGT1A6. MSU38225 strikingly decreases the protein level of Nrf2, which can be blocked by the proteasome inhibitor MG132. Ubiquitination of Nrf2 is enhanced following treatment with MSU38225. By inhibiting production of antioxidants, MSU38225 increases the level of reactive oxygen species (ROS) when cells are stimulated with tert-butyl hydroperoxide (tBHP). MSU38225 also inhibits the growth of human lung cancer cells in both two-dimensional cell culture and soft agar. Cancer cells addicted to Nrf2 are more susceptible to MSU38225 for suppression of cell proliferation. MSU38225 also sensitizes human lung cancer cells to chemotherapies both in vitro and in vivo Our results suggest that MSU38225 is a novel Nrf2 pathway inhibitor that could potentially serve as an adjuvant therapy to enhance the response to chemotherapies in patients with lung cancer.

Sustained, local delivery of the PARP inhibitor talazoparib prevents the development of mammary gland hyperplasia in Brca1-deficient mice.

Mutations in BRCA genes are the leading cause of hereditary breast cancer. Current options to prevent cancer in these high-risk patients, such as anti-estrogen drugs and radical mastectomy, are limited by lack of efficacy, undesirable toxicities, or physical and emotional challenges. We have previously shown that PARP inhibitors can significantly delay tumor development in BRCA1-deficient mice. Here, we fabricated the PARP inhibitor talazoparib (TLZ) into spacer implants (InCeT-TLZ) for localized and sustained delivery. We hypothesized that this novel formulation will provide an effective chemopreventive strategy with minimal toxicity. TLZ was released gradually over 30 days as implants degraded. InCeT-TLZ significantly decreased proliferation and increased DNA damage in the mammary glands of BRCA1-deficient mice. Notably, the number of mice that developed hyperplasia in the mammary glands was significantly lower with InCeT-TLZ treatment compared to the control group. Meanwhile, InCeT-TLZ was also better tolerated than oral TLZ, without loss of body weight or anemia. This study provides proof of concept for a novel and safe chemopreventive strategy using localized delivery of a PARP inhibitor for high-risk individuals. Future studies will directly evaluate the effects of InCeT-TLZ for preventing tumor development.

Western blot analysis of BK channel ß1-subunit expression should be interpreted cautiously when using commercially available antibodies.

Large conductance Ca(2+)-activated K(+) (BK) channels consist of pore-forming α- and accessory ß-subunits. There are four ß-subunit subtypes (ß1-ß4), BK ß1-subunit is specific for smooth muscle cells (SMC). Reduced BK ß1-subunit expression is associated with SMC dysfunction in animal models of human disease, because downregulation of BK ß1-subunit reduces channel activity and increases SMC contractility. Several anti-BK ß1-subunit antibodies are commercially available; however, the specificity of most antibodies has not been tested or confirmed in the tissues from BK ß1-subunit knockout (KO) mice. In this study, we tested the specificity and sensitivity of six commercially available antibodies from five manufacturers. We performed western blot analysis on BK ß1-subunit enriched tissues (mesenteric arteries and colons) and non-SM tissue (cortex of kidney) from wild-type (WT) and BK ß1-KO mice. We found that antibodies either detected protein bands of the appropriate molecular weight in tissues from both WT and BK ß1-KO mice or failed to detect protein bands at the appropriate molecular weight in tissues from WT mice, suggesting that these antibodies may lack specificity for the BK ß1-subunit. The absence of BK ß1-subunit mRNA expression in arteries, colons, and kidneys from BK ß1-KO mice was confirmed by RT-PCR analysis. We conclude that these commercially available antibodies might not be reliable tools for studying BK ß1-subunit expression in murine tissues under the denaturing conditions that we have used. Data obtained using commercially available antibodies should be interpreted cautiously. Our studies underscore the importance of proper negative controls in western blot analyses.