Endocardial cells form the coronary arteries by angiogenesis through myocardial-endocardial VEGF signaling.

The origins and developmental mechanisms of coronary arteries are incompletely understood. We show here by fate mapping, clonal analysis, and immunohistochemistry that endocardial cells generate the endothelium of coronary arteries. Dye tracking, live imaging, and tissue transplantation also revealed that ventricular endocardial cells are not terminally differentiated; instead, they are angiogenic and form coronary endothelial networks. Myocardial Vegf-a or endocardial Vegfr-2 deletion inhibited coronary angiogenesis and arterial formation by ventricular endocardial cells. In contrast, lineage and knockout studies showed that endocardial cells make a small contribution to the coronary veins, the formation of which is independent of myocardial-to-endocardial Vegf signaling. Thus, contrary to the current view of a common source for the coronary vessels, our findings indicate that the coronary arteries and veins have distinct origins and are formed by different mechanisms. This information may help develop better cell therapies for coronary artery disease.

ATM-CHK2-Beclin 1 axis promotes autophagy to maintain ROS homeostasis under oxidative stress.

The homeostatic link between oxidative stress and autophagy plays an important role in cellular responses to a wide variety of physiological and pathological conditions. However, the regulatory pathway and outcomes remain incompletely understood. Here, we show that reactive oxygen species (ROS) function as signaling molecules that regulate autophagy through ataxia-telangiectasia mutated (ATM) and cell cycle checkpoint kinase 2 (CHK2), a DNA damage response (DDR) pathway activated during metabolic and hypoxic stress. We report that CHK2 binds to and phosphorylates Beclin 1 at Ser90/Ser93, thereby impairing Beclin 1-Bcl-2 autophagy-regulatory complex formation in a ROS-dependent fashion. We further demonstrate that CHK2-mediated autophagy has an unexpected role in reducing ROS levels via the removal of damaged mitochondria, which is required for cell survival under stress conditions. Finally, CHK2-/- mice display aggravated infarct phenotypes and reduced Beclin 1 p-Ser90/Ser93 in a cerebral stroke model, suggesting an in vivo role of CHK2-induced autophagy in cell survival. Taken together, these results indicate that the ROS-ATM-CHK2-Beclin 1-autophagy axis serves as a physiological adaptation pathway that protects cells exposed to pathological conditions from stress-induced tissue damage.

Control of mitochondrial function and cell growth by the atypical cadherin Fat1.

Mitochondrial products such as ATP, reactive oxygen species, and aspartate are key regulators of cellular metabolism and growth. Abnormal mitochondrial function compromises integrated growth-related processes such as development and tissue repair, as well as homeostatic mechanisms that counteract ageing and neurodegeneration, cardiovascular disease, and cancer. Physiologic mechanisms that control mitochondrial activity in such settings remain incompletely understood. Here we show that the atypical Fat1 cadherin acts as a molecular 'brake' on mitochondrial respiration that regulates vascular smooth muscle cell (SMC) proliferation after arterial injury. Fragments of Fat1 accumulate in SMC mitochondria, and the Fat1 intracellular domain interacts with multiple mitochondrial proteins, including critical factors associated with the inner mitochondrial membrane. SMCs lacking Fat1 (Fat1KO) grow faster, consume more oxygen for ATP production, and contain more aspartate. Notably, expression in Fat1KO cells of a modified Fat1 intracellular domain that localizes exclusively to mitochondria largely normalizes oxygen consumption, and the growth advantage of these cells can be suppressed by inhibition of mitochondrial respiration, which suggest that a Fat1-mediated growth control mechanism is intrinsic to mitochondria. Consistent with this idea, Fat1 species associate with multiple respiratory complexes, and Fat1 deletion both increases the activity of complexes I and II and promotes the formation of complex-I-containing supercomplexes. In vivo, Fat1 is expressed in injured human and mouse arteries, and inactivation of SMC Fat1 in mice potentiates the response to vascular damage, with markedly increased medial hyperplasia and neointimal growth, and evidence of higher SMC mitochondrial respiration. These studies suggest that Fat1 controls mitochondrial activity to restrain cell growth during the reparative, proliferative state induced by vascular injury. Given recent reports linking Fat1 to cancer, abnormal kidney and muscle development, and neuropsychiatric disease, this Fat1 function may have importance in other settings of altered cell growth and metabolism.

Top-level leaders and implementation strategies to support organizational diversity, equity, inclusion, and belonging (DEIB) interventions: a qualitative study of top-level DEIB leaders in healthcare organizations.

BACKGROUND: The Black Lives Matter movement and COVID-19 pandemic motivated the wide-scale adoption of diversity, equity, inclusion, and belonging (DEIB) initiatives within healthcare organizations and the creation of DEIB top-level leader positions. The next step is to understand how these leaders contribute to the implementation of DEIB interventions, a task with notable salience due to not only the historical difficulties associated with DEIB strategy execution, but also the substantial evidence that leadership plays a significant role in implementation processes. Therefore, the objective of this qualitative study is to understand the role of top-level DEIB leaders in the implementation of healthcare organizational DEIB interventions. METHODS: A qualitative research approach which used an in-depth semi-structured interview approach was employed. We conducted thirty-one 60-90-min semi-structured interviews with DEIB top-level leaders between February 2022 and October 2022 over Zoom. An iterative coding process was used to identify the key implementation strategies and activities of DEIB top-level leaders. RESULTS: Interviewees were mostly Black, majority female, and mostly heterosexual and had a variety of educational backgrounds. We identified the DEIB top-level leader as the DEIB strategy implementation champion. These leaders drive five DEIB implementation strategies: (1) People, (2) Health Equity, (3) Monitoring and Feedback, (4) Operational Planning and Communication, and (5) External Partners. Within these, we identified 19 significant activities that describe the unique implementation strategies supported by the DEIB top-level leaders. CONCLUSIONS: To move toward sustained commitment to DEIB, the organization must focus on not only establishing DEIB interventions, but on their successful implementation. Our findings help explicate the implementation activities that drive the DEIB initiatives of healthcare organizations and the role of DEIB leaders. Our work can help healthcare organizations systematically identify how to support the success of DEIB organizational interventions.

Initiation of medication for opioid use disorder across a health system: A retrospective analysis of patient characteristics and inpatient outcomes.

Objectives: Medication for opioid use disorder (MOUD) has gained significant momentum as an evidence-based intervention for treating opioid use disorder (OUD). The purpose of this study was to characterize MOUD initiations for buprenorphine and extended release (ER) naltrexone across all care sites at a major health system in the Midwest and determine whether MOUD initiation was associated with inpatient outcomes. Methods: The study population comprised patients with OUD in the health system between 2018 and 2021. First, we described characteristics of all MOUD initiations for the study population within the health system. Second, we compared inpatient length of stay (LOS) and unplanned readmission rates between patients prescribed MOUD and patients not prescribed MOUD, including a pre-post comparison of patients prescribed MOUD before versus after initiation. Results: The 3,831 patients receiving MOUD were mostly white, non-Hispanic and generally received buprenorphine over ER naltrexone. 65.5% of most recent initiations occurred in an inpatient setting. Compared to those not prescribed MOUD, inpatient encounters where patients received MOUD on or before the admission date were significantly less likely to be unplanned readmissions (13% vs. 20%, p < 0.001) and their LOS was 0.14 days shorter (p = 0.278). Among patients prescribed MOUD, there was a significant reduction in the readmission rate after initiation compared to before (13% vs. 22%, p < 0.001). Conclusions: This study is the first to examine MOUD initiations for thousands of patients across multiple care sites in a health system, finding that receiving MOUD is associated with clinically meaningful reductions in readmission rates.

The deacetylation-phosphorylation regulation of SIRT2-SMC1A axis as a mechanism of antimitotic catastrophe in early tumorigenesis.

Improper distribution of chromosomes during mitosis can contribute to malignant transformation. Higher eukaryotes have evolved a mitotic catastrophe mechanism for eliminating mitosis-incompetent cells; however, the signaling cascade and its epigenetic regulation are poorly understood. Our analyses of human cancerous tissue revealed that the NAD-dependent deacetylase SIRT2 is up-regulated in early-stage carcinomas of various organs. Mass spectrometry analysis revealed that SIRT2 interacts with and deacetylates the structural maintenance of chromosomes protein 1 (SMC1A), which then promotes SMC1A phosphorylation to properly drive mitosis. We have further demonstrated that inhibition of SIRT2 activity or continuously increasing SMC1A-K579 acetylation causes abnormal chromosome segregation, which, in turn, induces mitotic catastrophe in cancer cells and enhances their vulnerability to chemotherapeutic agents. These findings suggest that regulation of the SIRT2-SMC1A axis through deacetylation-phosphorylation permits escape from mitotic catastrophe, thus allowing early precursor lesions to overcome oncogenic stress.

SIRT1 modulates cell cycle progression by regulating CHK2 acetylation-phosphorylation.

Both the stress-response protein, SIRT1, and the cell cycle checkpoint kinase, CHK2, play critical roles in aging and cancer via the modulation of cellular homeostasis and the maintenance of genomic integrity. However, the underlying mechanism linking the two pathways remains elusive. Here, we show that SIRT1 functions as a modifier of CHK2 in cell cycle control. Specifically, SIRT1 interacts with CHK2 and deacetylates it at lysine 520 residue, which suppresses CHK2 phosphorylation, dimerization, and thus activation. SIRT1 depletion induces CHK2 hyperactivation-mediated cell cycle arrest and subsequent cell death. In vivo, genetic deletion of Chk2 rescues the neonatal lethality of Sirt1-/- mice, consistent with the role of SIRT1 in preventing CHK2 hyperactivation. Together, these results suggest that CHK2 mediates the function of SIRT1 in cell cycle progression, and may provide new insights into modulating cellular homeostasis and maintaining genomic integrity in the prevention of aging and cancer.

Fidgetin-Like 2 siRNA Enhances the Wound Healing Capability of a Surfactant Polymer Dressing.

Microtubules (MTs) are intracellular polymers that provide structure to the cell, serve as railways for intracellular transport, and regulate many cellular activities, including cell migration. The dynamicity and function of the MT cytoskeleton are determined in large part by its regulatory proteins, including the recently discovered MT severing enzyme Fidgetin-like 2 (FL2). Downregulation of FL2 expression with small interfering RNA (siRNA) results in a more than twofold increase in cell migration rate in vitro as well as translates into improved wound-healing outcomes in in vivo mouse models. Here we utilized a commercially available surfactant polymer dressing (SPD) as a vehicle to deliver FL2 siRNA. To this end we incorporated collagen microparticles containing FL2 siRNA into SPD (SPD-FL2-siRNA) for direct application to the injury site. Topical application of SPD-FL2 siRNA to murine models of full-thickness excision wounds and full-thickness burn wounds resulted in significant improvements in the rate and quality of wound healing, as measured clinically and histologically, compared with controls. Wound healing occurred more rapidly and with high fidelity, resulting in properly organized collagen substructure. Taken together, these findings indicate that the incorporation of FL2 siRNA into existing treatment options is a promising avenue to improve wound outcomes.

Atg7 inhibits Warburg effect by suppressing PKM2 phosphorylation resulting reduced epithelial-mesenchymal transition.

Metabolic reprogramming is a distinct hallmark in tumorigenesis. Autophagy can rewire cell metabolism by regulating intracellular homeostasis. Warburg effect is a specific energy metabolic process that allows tumor cells to metabolize glucose via glycolysis into lactate even in the presence of oxygen. Although both autophagy and Warburg effect are involved in the stress response to energy crisis in tumor cells, their molecular relationship has remained largely elusive. We found that Atg7, a key molecule involved in autophagy, inhibits the Warburg effect. Mechanistically, Atg7 binds PKM2 and prevents its Tyr-105 phosphorylation by FGFR1. Furthermore, the hyperphosphorylation of PKM2 and its induced Warburg effect due to Atg7 deficiency promote epithelial-mesenchymal transition (EMT). Conversely, overexpression of Atg7 inhibits PKM2 phosphorylation and the Warburg effect, thereby inhibiting EMT of tumor cells. Our work reveals a molecular link between Atg7 and the Warburg effect, which may provide insight into novel strategies for cancer treatment.

Promotion and Suppression of Centriole Duplication Are Catalytically Coupled through PLK4 to Ensure Centriole Homeostasis.

PLK4 is the major kinase driving centriole duplication. Duplication occurs only once per cell cycle, forming one new (or daughter) centriole that is tightly engaged to the preexisting (or mother) centriole. Centriole engagement is known to block the reduplication of mother centrioles, but the molecular identity responsible for the block remains unclear. Here, we show that the centriolar cartwheel, the geometric scaffold for centriole assembly, forms the identity of daughter centrioles essential for the block, ceasing further duplication of the mother centriole to which it is engaged. To ensure a steady block, we found that the cartwheel requires constant maintenance by PLK4 through phosphorylation of the same substrate that drives centriole assembly, revealing a parsimonious control in which "assembly" and "block for new assembly" are linked through the same catalytic reaction to achieve homeostasis. Our results support a recently deduced model that the cartwheel-bound PLK4 directly suppresses centriole reduplication.

53BP1 and USP28 mediate p53-dependent cell cycle arrest in response to centrosome loss and prolonged mitosis.

Mitosis occurs efficiently, but when it is disturbed or delayed, p53-dependent cell death or senescence is often triggered after mitotic exit. To characterize this process, we conducted CRISPR-mediated loss-of-function screens using a cell-based assay in which mitosis is consistently disturbed by centrosome loss. We identified 53BP1 and USP28 as essential components acting upstream of p53, evoking p21-dependent cell cycle arrest in response not only to centrosome loss, but also to other distinct defects causing prolonged mitosis. Intriguingly, 53BP1 mediates p53 activation independently of its DNA repair activity, but requiring its interacting protein USP28 that can directly deubiquitinate p53 in vitro and ectopically stabilize p53 in vivo. Moreover, 53BP1 can transduce prolonged mitosis to cell cycle arrest independently of the spindle assembly checkpoint (SAC), suggesting that while SAC protects mitotic accuracy by slowing down mitosis, 53BP1 and USP28 function in parallel to select against disturbed or delayed mitosis, promoting mitotic efficiency.

Fidgetin-Like 2: A Microtubule-Based Regulator of Wound Healing.

Wound healing is a complex process driven largely by the migration of a variety of distinct cell types from the wound margin into the wound zone. In this study, we identify the previously uncharacterized microtubule-severing enzyme, Fidgetin-like 2 (FL2), as a fundamental regulator of cell migration that can be targeted in vivo using nanoparticle-encapsulated small interfering RNA (siRNA) to promote wound closure and regeneration. In vitro, depletion of FL2 from mammalian tissue culture cells results in a more than twofold increase in the rate of cell movement, in part due to a significant increase in directional motility. Immunofluorescence analyses indicate that FL2 normally localizes to the cell edge, importantly to the leading edge of polarized cells, where it regulates the organization and dynamics of the microtubule cytoskeleton. To clinically translate these findings, we utilized a nanoparticle-based siRNA delivery platform to locally deplete FL2 in both murine full-thickness excisional and burn wounds. Topical application of FL2 siRNA nanoparticles to either wound type results in a significant enhancement in the rate and quality of wound closure both clinically and histologically relative to controls. Taken together, these results identify FL2 as a promising therapeutic target to promote the regeneration and repair of cutaneous wounds.

Cep192 controls the balance of centrosome and non-centrosomal microtubules during interphase.

Cep192 is a centrosomal protein that contributes to the formation and function of the mitotic spindle in mammalian cells. Cep192's mitotic activities stem largely from its role in the recruitment to the centrosome of numerous additional proteins such as gamma-tubulin and Pericentrin. Here, we examine Cep192's function in interphase cells. Our data indicate that, as in mitosis, Cep192 stimulates the nucleation of centrosomal microtubules thereby regulating the morphology of interphase microtubule arrays. Interestingly, however, cells lacking Cep192 remain capable of generating normal levels of MTs as the loss of centrosomal microtubules is augmented by MT nucleation from other sites, most notably the Golgi apparatus. The depletion of Cep192 results in a significant decrease in the level of centrosome-associated gamma-tubulin, likely explaining its impact on centrosome microtubule nucleation. However, in stark contrast to mitosis, Cep192 appears to maintain an antagonistic relationship with Pericentrin at interphase centrosomes. Interphase cells depleted of Cep192 display significantly higher levels of centrosome-associated Pericentrin while overexpression of Cep192 reduces the levels of centrosomal Pericentrin. Conversely, depletion of Pericentrin results in elevated levels of centrosomal Cep192 and enhances microtubule nucleation at centrosomes, at least during interphase. Finally, we show that depletion of Cep192 negatively impacts cell motility and alters normal cell polarization. Our current working hypothesis is that the microtubule nucleating capacity of the interphase centrosome is determined by an antagonistic balance of Cep192, which promotes nucleation, and Pericentrin, which inhibits nucleation. This in turn determines the relative abundance of centrosomal and non-centrosomal microtubules that tune cell movement and shape.

Microtubule dynamics: Patronin, protector of the minus end.

It has long been surmised that cellular microtubules are capped at the minus ends to prevent their depolymerization. A recent study provides the first definitive identification of a minus-end-specific capping protein, termed Patronin, which protects the microtubule arrays of both mitotic and interphase cells.