LepR+ niche cell-derived AREG compromises hematopoietic stem cell maintenance under conditions of DNA repair deficiency and aging.

The cross talk between extrinsic niche-derived and intrinsic hematopoietic stem cell (HSC) factors controlling HSC maintenance remains elusive. Here, we demonstrated that amphiregulin (AREG) from bone marrow (BM) leptin receptor (LepR+) niche cells is an important factor that mediates the cross talk between the BM niche and HSCs in stem cell maintenance. Mice deficient of the DNA repair gene Brca2, specifically in LepR+ cells (LepR-Cre;Brca2fl/fl), exhibited increased frequencies of total and myeloid-biased HSCs. Furthermore, HSCs from LepR-Cre;Brca2fl/fl mice showed compromised repopulation, increased expansion of donor-derived, myeloid-biased HSCs, and increased myeloid output. Brca2-deficient BM LepR+ cells exhibited persistent DNA damage-inducible overproduction of AREG. Ex vivo treatment of wild-type HSCs or systemic treatment of C57BL/6 mice with recombinant AREG impaired repopulation, leading to HSC exhaustion. Conversely, inhibition of AREG by an anti-AREG-neutralizing antibody or deletion of the Areg gene in LepR-Cre;Brca2fl/fl mice rescued HSC defects caused by AREG. Mechanistically, AREG activated the phosphoinositide 3-kinases (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, promoted HSC cycling, and compromised HSC quiescence. Finally, we demonstrated that BM LepR+ niche cells from other DNA repair-deficient and aged mice also showed persistent DNA damage-associated overexpression of AREG, which exerts similar negative effects on HSC maintenance. Therefore, we identified an important factor that regulates HSCs function under conditions of DNA repair deficiency and aging.

Persistent DNA damage and oncogenic stress-induced Trem1 promotes leukemia in mice.

The immune receptor TREM1 (Triggering receptor expressed on myeloid cells 1) is a master regulator of inflammatory response. Compelling evidence suggests important pathological roles for TREM1 in various types of solid tumors. However, the role of TREM1 in hematologic malignancies is not known. Our previous study demonstrated that TREM1 cooperates with diminished DNA damage response to induce expansion of pre-leukemic hematopoietic stem cells (HSC) in mice deficient for the Fanconi anemia gene Fanca. Here we investigated TREM1 in leukemogenesis using mouse models of the DNA repair-deficient Fanca-/- and the oncogenic MLL-AF9 or KrasG12D. We found that Trem1 was highly expressed in preleukemic HSC and leukemia stem cells (LSC). By selective deletion of the Trem1 gene in the hematopoietic compartment, we showed that ablation of Trem1 reduced leukemogenic activity of the pre-leukemic HSC and LSC in mice. Trem1 was required for the proliferation of the pre-leukemic HSC and LSC. Further analysis revealed that Trem1 expression in preleukemic HSC and LSC was associated with persistent DNA damage, prolonged oncogenic stress, and a strong inflammatory signature. Targeting several top Trem1 inflammatory signatures inhibited the proliferation of pre-leukemic HSC and LSC. Collectively, our observations uncover previously unknown expression and function of TREM1 in malignant stem cells, and identify TREM1 as a driver of leukemogenesis.

Endothelial protection in lung grafts through heparanase inhibition during ex vivo lung perfusion in rats.

BACKGROUND: We hypothesized that enhancing glycocalyx preservation would reduce endothelial damage in lung grafts during ex-vivo lung perfusion (EVLP) leading to better transplant outcomes. In this study, we characterized the effects of inhibiting heparanase (HPSE), an enzyme responsible for glycocalyx shedding, on lung quality during EVLP. METHODS: Human clinical EVLP perfusate from lung graft patients was utilized to identify a potential association between glycocalyx integrity in grafted lung tissue and clinical data. In addition, we performed pre-clinical studies in which rat lungs underwent normothermic EVLP for 4 hours with/without HPSE inhibitors, heparin (1,000-U/h) or heparastatin (SF4; 1-µM), added to the perfusate. After 4-hours EVLP, left lungs were transplanted into syngeneic rats then evaluated for graft quality 2-hours after reperfusion. RESULTS: Clinically, increased degradation of syndecan-1 was identified in dysfunctional grafts during EVLP. Levels of heparan sulfate in perfusate after EVLP were associated with incidence of graft dysfunction after transplantation. In the pre-clinical rat study, SF4 effectively inhibited HPSE activity, and significantly attenuated dissociated glycocalyx levels, endothelial dysfunction, edema, and inflammation in lungs during EVLP compared to both controls and heparin groups. High-doses of heparin demonstrated markedly increased perfusate syndecan-1 concentrations and deteriorated lung quality during EVLP compared with controls. Post-transplant graft function and inflammation were significantly improved in SF4-treated group compared to those in both control and heparin-treated groups. CONCLUSIONS: This study demonstrated that HPSE activity inhibition by SF4 can improve graft preservation during EVLP by protecting the glycocalyx and endothelial function, leading to better lung function following transplantation.

Impaired Cellular and Antibody immunity after COVID-19 in Chronically Immunosuppressed Transplant Recipients.

Assessment of cellular immunity to the SARS-CoV-2 coronavirus is of great interest in chronically immunosuppressed transplant recipients (Tr), who are predisposed to infections and vaccination failures. We evaluated CD154-expressing T-cells induced by spike (S) antigenic peptides in 204 subjects-103 COVID-19 patients and 101 healthy unexposed subjects. S-reactive CD154+T-cell frequencies were a) higher in 42 healthy unexposed Tr who were sampled pre-pandemic, compared with healthy NT (p=0.02), b) lower in Tr COVID-19 patients compared with healthy Tr (p<0.0001) and were accompanied by lower S-reactive B-cell frequencies (p<0.05), c) lower in Tr with severe COVID-19 (p<0.0001), or COVID-19 requiring hospitalization (p<0.05), compared with healthy Tr. Among Tr with COVID-19, cytomegalovirus co-infection occurred in 34%; further, incidence of anti-receptor-binding-domain IgG (p=0.011) was lower compared with NT COVID-19 patients. Healthy unexposed Tr exhibit pre-existing T-cell immunity to SARS-CoV-2. COVID-19 impairs anti-S T-cell and antibody and predisposes to CMV co-infection in transplant recipients.

Hematopoietic stem cell regeneration through paracrine regulation of the Wnt5a/Prox1 signaling axis.

The crosstalk between the BM microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration. Here, we show that in mice, deletion of the Fanconi anemia (FA) genes Fanca and Fancc dampened HSC regeneration through direct effects on HSCs and indirect effects on BM niche cells. FA HSCs showed persistent upregulation of the Wnt target Prox1 in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improved long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in WT HSCs mimicked the defective repopulation phenotype of FA HSCs. WT mice but not FA mice showed significant induction by TBI of BM stromal Wnt5a protein. Mechanistically, FA proteins regulated stromal Wnt5a expression, possibly through modulating the Wnt5a transcription activator Pax2. Wnt5a treatment of irradiated FA mice enhanced HSC regeneration. Conversely, Wnt5a neutralization inhibited HSC regeneration after TBI. Wnt5a secreted by LepR+CXCL12+ BM stromal cells inhibited ß-catenin accumulation, thereby repressing Prox1 transcription in irradiated HSCs. The detrimental effect of deregulated Wnt5a/Prox1 signaling on HSC regeneration was also observed in patients with FA and aged mice. Irradiation induced upregulation of Prox1 in the HSCs of aged mice, and deletion of Prox1 in aged HSCs improved HSC regeneration. Treatment of aged mice with Wnt5a enhanced hematopoietic repopulation. Collectively, these findings identified the paracrine Wnt5a/Prox1 signaling axis as a regulator of HSC regeneration under conditions of injury and aging.

Matrixmetalloproteinase Inhibitors: Promising Therapeutic Targets Against Cancer.

BACKGROUND: Cancer is a wide range cellular level disease that occurs when cells go through uncontrolled division and growth. The mechanisms by which the cells undergo metastasis are complex and involve many interactions between the tumor cells and their cellular environment. Matrix metalloproteinases (MMPs) have been found to over-express at various stages of tumor progression and their inhibition using MMP inhibitors has been a subject of potential therapy against cancer. OBJECTIVE: This review discusses recent research in MMP inhibitors (MMPI) used for preventing tumor progression. METHODS: In this review, we explored the role of MMPs in cancer progression and summarized the current developments in MMPIs, their role in cancer suppression in in vitro and in vivo studies and their evaluation in clinical trials from the current research data. RESULTS: MMPIs have shown to be very successful in in vitro models, cell lines and in some in vivo studies. Unfortunately, their efficacy in clinical trials has been found to be hit and miss. Recent studies have shown that the novel delivery approaches of MMP inhibitors may enhance their therapeutic effects towards the prevention of cancer. CONCLUSION: In this review, we presented different MMP inhibitors, their performance at different stages of models - in vitro, in vivo, small animal models and eventually clinical trials. We provide newer methods of MMPI delivery that may be better targeted to suppress only specific MMPs and avoid toxic side effects in healthy cells.

Human Gut Microbiome: A New Frontier in Cancer Diagnostics & Therapeutics.

The field of oncology is vast and ever-growing. The present cancer therapeutics is continually exhibiting various drawbacks, which opens the door for exploring better novel therapeutic techniques. One such emerging technique is the manipulation of gut microbiota to induce a positive curative effect in the body. The dynamic gut microbiota of our body houses an astonishing number of microorganisms, mainly bacteria. The balance of the gut microbiota is essential for good health as imbalances may result in dysbiosis leading to various diseases such as cancer. The gut microbiota can be manipulated by using prebiotics, probiotics, synbiotics, postbiotics, and antibiotics for better therapeutic outcomes, as well as to improve the quality of life of patients undergoing conventional cancer treatment. Administration of bacteria as a probiotic agent accompanied with prebiotics obtained from a wide variety of herbs has been used effectively to enhance the treatment of various cancers. Although the theoretical basis of Gut therapy can be ascertained, further clinical trials will be essential to determine the scope and limitations fully. The present review provides a glimpse of conventional and novel cancer therapeutics and their drawbacks, along with the role of the gut microbiome and its modulation to design new pharmaceutics against cancer.

Anti-inflammatory Effects of S. cumini Seed Extract on Gelatinase-B (MMP-9) Regulation against Hyperglycemic Cardiomyocyte Stress.

Black berry (Syzygium cumini) fruit is useful in curing diabetic complications; however, its role in diabetes-induced cardiomyopathy is not yet known. In this study, we investigated the regulation of gelatinase-B (MMP-9) by S. cumini methanol seed extract (MSE) in diabetic cardiomyopathy using real-time PCR, RT-PCR, immunocytochemistry, gel diffusion assay, and substrate zymography. The regulatory effects of MSE on NF-κB, TNF-α, and IL-6 were also examined. Identification and estimation of polyphenol constituents present in S. cumini extract were carried out using reverse-phase HPLC. Further, in silico docking studies of identified polyphenols with gelatinase-B were performed to elucidate molecular level interaction in the active site of gelatinase-B. Docking studies showed strong interaction of S. cumini polyphenols with gelatinase-B. Our findings indicate that MSE significantly suppresses gelatinase-B expression and activity in high-glucose- (HG-) stimulated cardiomyopathy. Further, HG-induced activation of NF-κB, TNF-α, and IL-6 was also remarkably reduced by MSE. Our results suggest that S. cumini MSE may be useful as an effective functional food and dietary supplement to regulate HG-induced cardiac stress through gelatinase.

Impaired T-cell and antibody immunity after COVID-19 infection in chronically immunosuppressed transplant recipients.

Assessment of T-cell immunity to the COVID-19 coronavirus requires reliable assays and is of great interest, given the uncertain longevity of the antibody response. Some recent reports have used immunodominant spike (S) antigenic peptides and anti-CD28 co-stimulation in varying combinations to assess T-cell immunity to SARS-CoV-2. These assays may cause T-cell hyperstimulation and could overestimate antiviral immunity in chronically immunosuppressed transplant recipients, who are predisposed to infections and vaccination failures. Here, we evaluate CD154-expressing T-cells induced by unselected S antigenic peptides in 204 subjects-103 COVID-19 patients and 101 healthy unexposed subjects. Subjects included 72 transplanted and 130 non-transplanted subjects. S-reactive CD154+T-cells co-express and can thus substitute for IFNγ (n=3). Assay reproducibility in a variety of conditions was acceptable with coefficient of variation of 2-10.6%. S-reactive CD154+T-cell frequencies were a) higher in 42 healthy unexposed transplant recipients who were sampled pre-pandemic, compared with 59 healthy non-transplanted subjects (p=0.02), b) lower in Tr COVID-19 patients compared with healthy transplant patients (p<0.0001), c) lower in Tr patients with severe COVID-19 (p<0.0001), or COVID-19 requiring hospitalization (p<0.05), compared with healthy Tr recipients. S-reactive T-cells were not significantly different between the various COVID-19 disease categories in NT recipients. Among transplant recipients with COVID-19, cytomegalovirus co-infection occurred in 34%; further, CMV-specific T-cells (p<0.001) and incidence of anti-receptor-binding-domain IgG (p=0.011) were lower compared with non-transplanted COVID-19 patients. Healthy unexposed transplant recipients exhibit pre-existing T-cell immunity to SARS-CoV-2. COVID-19 infection leads to impaired T-cell and antibody responses to SARS-CoV-2 and increased risk of CMV co-infection in transplant recipients.

Pathophysiology, Clinical Characteristics of Diabetic Cardiomyopathy: Therapeutic Potential of Natural Polyphenols.

Diabetic cardiomyopathy (DCM) is an outcome of disturbances in metabolic activities through oxidative stress, local inflammation, and fibrosis, as well as a prime cause of fatality worldwide. Cardiovascular disorders in diabetic individuals have become a challenge in diagnosis and formulation of treatment prototype. It is necessary to have a better understanding of cellular pathophysiology that reveal the therapeutic targets and prevent the progression of cardiovascular diseases due to hyperglycemia. Critical changes in levels of collagen and integrin have been observed in the extracellular matrix of heart, which was responsible for cardiac remodeling in diabetic patients. This review explored the understanding of the mechanisms of how the phytochemicals provide cardioprotection under diabetes along with the caveats and provide future perspectives on these agents as prototypes for the development of drugs for managing DCM. Thus, here we summarized the effect of various plant extracts and natural polyphenols tested in preclinical and cell culture models of diabetic cardiomyopathy. Further, the potential use of selected polyphenols that improved the therapeutic efficacy against diabetic cardiomyopathy is also illustrated.

Synthesis and Characterization of Sygyzium cumini Nanoparticles for Its Protective Potential in High Glucose-Induced Cardiac Stress: a Green Approach.

There exists a complex and multifactorial relationship between diabetes and cardiovascular disease. Hyperglycemia is an important factor imposing damage (glucose toxicity) on cardiac cell leading to diabetic cardiomyopathy. There are substantial clinical evidences on the adverse effects of conventional therapies in the prevention/treatment of diabetic cardiovascular complications. Currently, green-synthesized nanoparticles have emerged as a safe, efficient, and inexpensive alternative for therapeutic uses. The present study discloses the silver nanoparticle biosynthesizing capability and cardioprotective potential of Syzygium cumini seeds already reported to have antidiabetic properties. Newly generated silver nanoparticles S. cumini MSE silver nanoparticles (SmSNPs) were characterized by UV-visible spectroscopy, scanning electron microscopy (SEM), zeta sizer, X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. Using methanolic extract of S. cumini seeds, an average size of 40-100-nm nanoparticles with 43.02 nm and -19.6 mV zeta potential were synthesized. The crystalline nature of SmSNPs was identified by using XRD. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) assays revealed the antioxidative potential to be 66.87 (±0.7) % and 86.07 (±0.92) % compared to 60.29 (±0.02) % and 85.67 (±1.27) % for S. cumini MSE. In vitro study on glucose-stressed H9C2 cardiac cells showed restoration in cell size, nuclear morphology, and lipid peroxide formation upon treatment of SmSNPs. Our findings concluded that S. cumini MSE SmSNPs significantly suppress the glucose-induced cardiac stress in vitro by maintaining the cellular integrity and reducing the oxidative damages therefore establishing its therapeutic potential in diabetic cardiomyopathy.

An assessment of norepinephrine mediated hypertrophy to apoptosis transition in cardiac cells: a signal for cell death.

OBJECTIVES: Heart is an organ which is under a constant work load that generates numerous stress responses. Heart failure is associated with increased plasma norepinephrine (NE) and hypertrophic cell death. Within the current study we try to understand the concentration dependent molecular switch from hypertrophy to apoptosis under stress. METHODS: The effect of increasing concentration of NE on cell death was studied using MTT assay based on which further experimental conditions were decided. Trypan Blue staining and TUNEL assay were done at selected concentrations of NE. Cellular and nuclear morphology at these concentrations was studied using Haematoxylin-Eosin, DAPI and PI stains. The molecular switch between hypertrophy and cell death was studied by expression analysis of ß-MyHC and TNF-α. Rhodamine and DCFH-DA staining were done to evaluate the role of mitochondria and ROS under these conditions. Role of caspases under these transitions was also evaluated. RESULT: NE shows steep falls in cell viability at 50 µM and 100 µM concentrations. The cellular and nuclear morphology is altered at these concentrations along with alterations at molecular level showing a shift from hypertrophy towards cell death. Altered mitochondrial membrane potential and increase in ROS support this which leads to caspase dependent activation of cell death. CONCLUSION: We show that at 50 µM NE, there occurs a transition from cellular hypertrophy towards death. This could be beneficial to prevent hypertrophy induced cardiac cell death and evaluating cardio protective therapeutic targets in vitro.

Protective effect of Syzygium cumini against pesticide-induced cardiotoxicity.

Pesticide-induced toxicity is a serious issue which has resulted in plethora of diseases all over the world. The organophosphate pesticide malathion has caused many incidents of poisoning such as cardiac manifestations. The present study was designed to evaluate the effect of Syzygium cumini on malathion-induced cardiotoxicity. Dose optimization of malathion and polyphenols such as curcumin, (−)-epicatechin, gallic acid, butylated hydroxyl toluene, etc. was done by MTT cell proliferation assay. Nuclear deformities, ROS production, and integrity of extra cellular matrix components were analyzed by different techniques. S. cumini methanolic pulp extract (MPE), a naturally derived gallic acid-enriched antioxidant was taken to study its effect on malathion-induced toxicity. Nuclear deformities, ROS production, and integrity of extra cellular matrix components were also analyzed. Twenty micrograms per milliliter LD50 dose of malathion was found to cause stress-mediated responses in H9C2 cell line. Among all the polyphenols, gallic acid showed the most significant protection against stress. Gallic acid-enriched methanolic S. cumini pulp extract (MPE) showed 59.76 % ± 0.05, 81.61 % ± 1.37, 73.33 % ± 1.33, 77.19 % ± 2.38 and 64.19 % ± 1.43 maximum inhibition for DPPH, ABTS, NO, H2O2 and superoxide ion, respectively, as compared to ethanolic pulp extract and aqueous pulp extract. Our study suggests that S. cumini MPE has the ability to protect against the malathion-mediated oxidative stress in cardiac myocytes.

Cardioprotective role of Syzygium cumini against glucose-induced oxidative stress in H9C2 cardiac myocytes.

Diabetic patients are known to have an independent risk of cardiomyopathy. Hyperglycemia leads to upregulation of reactive oxygen species (ROS) that may contribute to diabetic cardiomyopathy. Thus, agents that suppress glucose-induced intracellular ROS levels can have therapeutic potential against diabetic cardiomyopathy. Syzygium cumini is well known for its anti-diabetic potential, but its cardioprotective properties have not been evaluated yet. The aim of the present study is to analyze cardioprotective properties of methanolic seed extract (MSE) of S. cumini in diabetic in vitro conditions. ROS scavenging activity of MSE was studied in glucose-stressed H9C2 cardiac myoblasts after optimizing the safe dose of glucose and MSE by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide. 2',7'-dichlorfluorescein diacetate staining and Fluorescence-activated cell sorting analysis confirmed the suppression of ROS production by MSE in glucose-induced cells. The intracellular NO and H2O2 radical-scavenging activity of MSE was found to be significantly high in glucose-induced cells. Exposure of glucose-stressed H9C2 cells to MSE showed decline in the activity of catalase and superoxide dismutase enzymes and collagen content. 4',6-diamidino-2-phenylindole, propidium iodide and 10-N-nonyl-3,6-bis (dimethylamino) acridine staining revealed that MSE protects myocardial cells from glucose-induced stress. Taken together, our findings revealed that the well-known anti-diabetic S. cumini can also protect the cardiac cells from glucose-induced stress.