REDD1 Deletion Suppresses NF-κB Signaling in Cardiomyocytes and Prevents Deficits in Cardiac Function in Diabetic Mice.

Activation of the transcription factor NF-κB in cardiomyocytes has been implicated in the development of cardiac function deficits caused by diabetes. NF-κB controls the expression of an array of pro-inflammatory cytokines and chemokines. We recently discovered that the stress response protein regulated in development and DNA damage response 1 (REDD1) was required for increased pro-inflammatory cytokine expression in the hearts of diabetic mice. The studies herein were designed to extend the prior report by investigating the role of REDD1 in NF-κB signaling in cardiomyocytes. REDD1 genetic deletion suppressed NF-κB signaling and nuclear localization of the transcription factor in human AC16 cardiomyocyte cultures exposed to TNFα or hyperglycemic conditions. A similar suppressive effect on NF-κB activation and pro-inflammatory cytokine expression was also seen in cardiomyocytes by knocking down the expression of GSK3ß. NF-κB activity was restored in REDD1-deficient cardiomyocytes exposed to hyperglycemic conditions by expression of a constitutively active GSK3ß variant. In the hearts of diabetic mice, REDD1 was required for reduced inhibitory phosphorylation of GSK3ß at S9 and upregulation of IL-1ß and CCL2. Diabetic REDD1+/+ mice developed systolic functional deficits evidenced by reduced ejection fraction. By contrast, REDD1-/- mice did not exhibit a diabetes-induced deficit in ejection fraction and left ventricular chamber dilatation was reduced in diabetic REDD1-/- mice, as compared to diabetic REDD1+/+ mice. Overall, the results support a role for REDD1 in promoting GSK3ß-dependent NF-κB signaling in cardiomyocytes and in the development of cardiac function deficits in diabetic mice.

Defibrotide mitigates endothelial cell injury induced by plasmas from patients with COVID-19 and related vasculopathies.

BACKGROUND AND OBJECTIVES: COVID-19 progression is characterized by systemic small vessel arterial and venous thrombosis. Microvascular endothelial cell (MVEC) activation and injury, platelet activation, and histopathologic features characteristic of acute COVID-19 also describe certain thrombotic microangiopathies, including atypical hemolytic-uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), and hematopoietic stem cell transplant (HSCT)-associated veno-occlusive disease (VOD). We explored the effect of clinically relevant doses of defibrotide, approved for HSCT-associated VOD, on MVEC activation/injury. METHODS: Human dermal MVEC were exposed to plasmas from patients with acute TMAs or acute COVID-19 in the presence and absence of defibrotide (5µg/ml) and caspase 8, a marker of EC activation and apoptosis, was assessed. RNAseq was used to explore potential mechanisms of defibrotide activity. RESULTS: Defibrotide suppressed TMA plasma-induced caspase 8 activation in MVEC (mean 60.2 % inhibition for COVID-19; p = 0.0008). RNAseq identified six major cellular pathways associated with defibrotide's alteration of COVID-19-associated MVEC changes: TNF-α signaling; IL-17 signaling; extracellular matrix (ECM)-EC receptor and platelet receptor interactions; ECM formation; endothelin activity; and fibrosis. Communications across these pathways were revealed by STRING analyses. Forty transcripts showing the greatest changes induced by defibrotide in COVID-19 plasma/MVEC cultures included: claudin 14 and F11R (JAM), important in maintaining EC tight junctions; SOCS3 and TNFRSF18, involved in suppression of inflammation; RAMP3 and transgelin, which promote angiogenesis; and RGS5, which regulates caspase activation and apoptosis. CONCLUSION: Our data, in the context of a recent clinical trial in severe COVID-19, suggest benefits to further exploration of defibrotide and these pathways in COVID-19 and related endotheliopathies.

Hyperphagia in offsprings of in utero hyperglycemic mothers is associated with increased expression of heparan sulfate proteoglycans in hypothalamus.

In utero hyperglycemia has consequences on future outcomes in the offsprings. We had earlier shown that in utero hyperglycemia impacts proteoglycans/glycosaminoglycans, one of the key molecules involved in brain development. Hypothalamic HSPGs such as syndecan-1 and syndecan-3 are well known for their involvement in feeding behavior. Therefore, studies were carried out to determine the effect of maternal hyperglycemia on the expression of HSPGs in the hypothalamus of offspring brain. Results revealed increased protein abundance of Syndecan-1 and -3 as well as glypican-1 in postnatal adults from hyperglycemic mothers. This was associated with increased hyperphagia and increased expression of Neuropeptide Y. These results indicate the likely consequences on offsprings exposed to in utero hyperglycemia on its growth.

Megakaryocyte/platelet-derived TGF-ß1 inhibits megakaryopoiesis in bone marrow by regulating thrombopoietin production in liver.

Transforming growth factor ß1 (TGF-ß1) regulates a wide variety of events in adult bone marrow (BM), including quiescence of hematopoietic stem cells, via undefined mechanisms. Because megakaryocytes (MKs)/platelets are a rich source of TGF-ß1, we assessed whether TGF-ß1 might inhibit its own production by comparing mice with conditional inactivation of Tgfb1 in MKs (PF4Cre;Tgfb1flox/flox) and control mice. PF4Cre;Tgfb1flox/flox mice had ∼30% more MKs in BM and ∼15% more circulating platelets than control mice (P < .001). Thrombopoietin (TPO) levels in plasma and TPO expression in liver were approximately twofold higher in PF4Cre;Tgfb1flox/flox than in control mice (P < .01), whereas TPO expression in BM cells was similar between these mice. In BM cell culture, TPO treatment increased the number of MKs from wild-type mice by approximately threefold, which increased approximately twofold further in the presence of a TGF-ß1-neutralizing antibody and increased the number of MKs from PF4Cre;Tgfb1flox/flox mice approximately fourfold. Our data reveal a new role for TGF-ß1 produced by MKs/platelets in regulating its own production in BM via increased TPO production in the liver. Additional studies are required to determine the mechanism.