CK2ß Regulates Hematopoietic Stem Cell Biology and Erythropoiesis.

The Ser-Thr kinase CK2 plays important roles in sustaining cell survival and resistance to stress and these functions are exploited by different types of blood tumors. Yet, the physiological involvement of CK2 in normal blood cell development is poorly known. Here, we discovered that the ß regulatory subunit of CK2 is critical for normal hematopoiesis in the mouse. Fetal livers of conditional CK2ß knockout embryos showed increased numbers of hematopoietic stem cells associated to a higher proliferation rate compared to control animals. Both hematopoietic stem and progenitor cells (HSPCs) displayed alterations in the expression of transcription factors involved in cell quiescence, self-renewal, and lineage commitment. HSPCs lacking CK2ß were functionally impaired in supporting both in vitro and in vivo hematopoiesis as demonstrated by transplantation assays. Furthermore, KO mice developed anemia due to a reduced number of mature erythroid cells. This compartment was characterized by dysplasia, proliferative defects at early precursor stage, and apoptosis at late-stage erythroblasts. Erythroid cells exhibited a marked compromise of signaling cascades downstream of the cKit and erythropoietin receptor, with a defective activation of ERK/JNK, JAK/STAT5, and PI3K/AKT pathways and perturbations of several transcriptional programs as demonstrated by RNA-Seq analysis. Moreover, we unraveled an unforeseen molecular mechanism whereby CK2 sustains GATA1 stability and transcriptional proficiency. Thus, our work demonstrates new and crucial functions of CK2 in HSPC biology and in erythropoiesis.

Post-Transcriptional and Epigenetic Regulation of Estrogen Signaling.

Post-translational and epigenetic regulation are important mechanisms controlling functions of genes and proteins. Although the "classic" estrogen receptors (ERs) have been acknowledged to function in mediating estrogen effects via transcriptional mechanisms, estrogenic agents modulate the turnover of several proteins via post-transcriptional and post-translational pathways including epigenetics. For instance, the metabolic and angiogenic action of G-protein coupled estrogen receptor (GPER) in vascular endothelial cells has been recently elucidated. By interacting with GPER, 17ß-estradiol and the GPER agonist G1 enhance endothelial stability of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and capillary tube formation by increasing ubiquitin-specific peptidase 19 levels, thereby reducing PFKFB3 ubiquitination and proteasomal degradation. In addition to ligands, the functional expression and trafficking of ERs can be modulated by post-translational modification, including palmitoylation. MicroRNAs (miRNAs), the most abundant form of endogenous small RNAs in humans, regulate multiple target genes and are at the center of the multi-target regulatory network. This review also discusses the emerging evidence of how miRNAs affect glycolytic metabolism in cancer, as well as their regulation by estrogens. Restoring dysregulated miRNA expression represents a promising strategy to counteract the progression of cancer and other disease conditions. Accordingly, estrogen post-transcriptional regulatory and epigenetic mechanisms represent novel targets for pharmacological and nonpharmacological intervention for the treatment and prevention of hormone-sensitive noncommunicable diseases, including estrogen-sensitive cancers of the reproductive system in women. SIGNIFICANCE STATEMENT: The effects of estrogen are mediated by several mechanisms that are not limited to the transcriptional regulation of target genes. Slowing down the turnover of master regulators of metabolism by estrogens allows cells to rapidly adapt to environmental cues. Identification of estrogen-targeted microRNAs may lead to the development of novel RNA therapeutics that disrupt pathological angiogenesis in estrogen-dependent cancers.

Evaluation of post-natal angiogenesis in a mouse hind limb ischemia model.

Hind limb ischemia is a useful model to assess metabolic and cellular responses. Here, we present a protocol for evaluating post-natal angiogenesis in a mouse hind limb ischemia model. We describe steps to induce a severe restriction of blood supply of the femoral artery and vein that mimics the real-life scenario observed in clinical settings. We then detail procedures for follow-up laser Doppler imaging to compare post-ischemic responses of four different mouse strains in their capacity to trigger compensatory arteriogenesis. For complete details on the use and execution of this protocol, please refer to Oberkersch et al. (2022).1.

Leucocyte Abnormalities in Synovial Fluid of Degenerative and Inflammatory Arthropathies.

Genome damage has been related to the induction of autoimmune processes, chronic inflammation, and apoptosis. Recent studies suggest that some rheumatological diseases are associated with overall genomic instability in the T cell compartment. However, no data regarding leucocyte abnormalities in synovial fluid (SF) and their relationship with inflammation are available. The aim of this study was to investigate cellular phenotypes in SF collected from patients with different inflammatory arthropathies, including rhematoid arthritis (RA), psoriatic arthritis (PsA), crystal-induced arthritis (CIA), and non-inflammatory arthropathies, such as osteoarthritis (OA). We found high percentage of micronuclei in SF from CIA compared to the other groups and a high frequency of pyknotic cell in RA and CIA patients. A correlation between pyknosis and immature polymorphonuclear cells with local inflammatory indices was observed. The study of the apoptosis process revealed an increased BAX expression in CIA and RA compared to OA and PsA, while Bcl-2 was higher in CIA. Caspase-3 activity was increased in SF from RA patients and correlates with inflammatory and anti-inflammatory cytokines. In conclusion, our results showed that inflammatory SF is associated with genomic instability and abnormal cell subsets.

Loss of Hematopoietic Cell-Derived Oncostatin M Worsens Diet-Induced Dysmetabolism in Mice.

Innate immune cells infiltrate growing adipose tissue and propagate inflammatory clues to metabolically distant tissues, thereby promoting glucose intolerance and insulin resistance. Cytokines of the IL-6 family and gp130 ligands are among such signals. The role played by oncostatin M (OSM) in the metabolic consequences of overfeeding is debated, at least in part, because prior studies did not distinguish OSM sources and dynamics. Here, we explored the role of OSM in metabolic responses and used bone marrow transplantation to test the hypothesis that hematopoietic cells are major contributors to the metabolic effects of OSM. We show that OSM is required to adapt during the development of obesity because OSM concentrations are dynamically modulated during high-fat diet (HFD) and Osm-/- mice displayed early-onset glucose intolerance, impaired muscle glucose uptake, and worsened liver inflammation and damage. We found that OSM is mostly produced by blood cells and deletion of OSM in hematopoietic cells phenocopied glucose intolerance of whole-body Osm-/- mice fed a HFD and recapitulated liver damage with increased aminotransferase levels. We thus uncovered that modulation of OSM is involved in the metabolic response to overfeeding and that hematopoietic cell-derived OSM can regulate metabolism, likely via multiple effects in different tissues.

Hematopoietic Stem Cells and Metabolic Deterioration in Alström Syndrome, a Rare Genetic Model of the Metabolic Syndrome.

Alström syndrome (AS) is a rare genetic disease caused by ALMS1 mutations, characterized by short stature, and vision and hearing loss. Patients with AS develop the metabolic syndrome, long-term organ complications, and die prematurely. We explored the association between AS and a shortage of hematopoietic stem/progenitor cells (HSPCs), which is linked to metabolic diseases and predicts diabetic complications. We included patients with AS at a national referral center. We measured HSPCs with flow cytometry at baseline and follow-up. We followed patients up to January 2022 for metabolic worsening and end-organ damage. We evaluated HSPC levels and mobilization as well as bone marrow histology in a murine model of AS. In 23 patients with AS, we found significantly lower circulating HSPCs than in healthy blood donors (-40%; P = .002) and age/sex-matched patients (-25%; P = .022). Longitudinally, HSPCs significantly declined by a further 20% in patients with AS over a median of 36 months (interquartile range 30-44). Patients with AS who displayed metabolic deterioration over 5.3 years had lower levels of HSPCs, both at baseline and at last observation, than those who did not deteriorate. Alms1-mutated mice were obese and insulin resistant and displayed significantly reduced circulating HSPCs, despite no overt hematological abnormality. Contrary to what was observed in diabetic mice, HSPC mobilization and bone marrow structure were unaffected. We found depletion of HSPCs in patients with AS, which was recapitulated in Alms1-mutated mice. Larger and longer studies will be needed to establish HSPCs shortage as a driver of metabolic deterioration leading to end-organ damage in AS.

Synovial Fluid from Patients with Osteoarthritis Shows Different Inflammatory Features Depending on the Presence of Calcium Pyrophosphate Crystals.

The role of calcium pyrophosphate (CPP) crystals in osteoarthritis (OA) is still a matter of debate. With this study we aimed to investigate the inflammatory features of synovial fluid (SF) collected from patients with OA with CPP crystals compared with those without crystals. We also explored the effect of OA SF on monocytes response. SFs were collected from adult patients with OA and subdivided according to the presence of crystals. Local cellular and humoral inflammatory mediators were analysed in the SF samples. The expression levels of IL-1ß, IL-18, CASP-1, NLRP3, and GAPDH were measured by RT-PCR in the cells obtained by pelleting the SF samples. For the in vitro study, a monocytic cell line was treated with selected SF samples. SF with CPP crystals showed a significant increase in inflammatory cellular indices and higher levels of IL-1ß, IL-8, and caspase-1 transcript with respect to SF without crystals. Higher concentrations of VEGF were also observed in the early stages of the whole OA patients. THP-1 cells stimulated with OA SF released a significant amount of IL-1 ß in culture supernatants. This study demonstrated that SF collected from patients with OA shows different inflammatory features depending on the presence of CPP crystals.

Mannose Receptor Deficiency Impacts Bone Marrow and Circulating Immune Cells during High Fat Diet Induced Obesity.

The mannose receptor C-type 1 (Mrc1) is a C-type lectin receptor expressed on the immune cells and sinusoidal endothelial cells (ECs) of several tissues, including the bone marrow (BM). Parallel to systemic metabolic alterations and hematopoietic cell proliferation, high-fat diet (HFD) feeding increases the expression of Mrc1 in sinusoidal ECs, thus calling for the investigation of its role in bone marrow cell reprogramming and the metabolic profile during obesity. Mrc1-/- mice and wild-type (WT) littermates were fed an HFD (45% Kcal/diet) for 20 weeks. Weight gain was monitored during the diet regimen and glucose and insulin tolerance were assessed. Extensive flow cytometry profiling, histological, and proteomic analyses were performed. After HFD feeding, Mrc1-/- mice presented impaired medullary hematopoiesis with reduced myeloid progenitors and mature cells in parallel with an increase in BM adipocytes compared to controls. Accordingly, circulating levels of neutrophils and pro-inflammatory monocytes decreased in Mrc1-/- mice together with reduced infiltration of macrophages in the visceral adipose tissue and the liver compared to controls. Liver histological profiling coupled with untargeted proteomic analysis revealed that Mrc1-/- mice presented decreased liver steatosis and the downregulation of proteins belonging to pathways involved in liver dysfunction. This profile was reflected by improved glucose and insulin response and reduced weight gain during HFD feeding in Mrc1-/- mice compared to controls. Our data show that during HFD feeding, mannose receptor deficiency impacts BM and circulating immune cell subsets, which is associated with reduced systemic inflammation and resistance to obesity development.

Semisolid Wet Sol-Gel Silica/Hydroxypropyl Methyl Cellulose Formulation for Slow Release of Serpin B3 Promotes Wound Healing In Vivo.

Foot ulcerations are a disabling complication of diabetes and no treatment is currently available based on disease mechanisms. The protein serpin B3 (SB3) was identified as a positive biomarker of successful diabetic wound healing; therefore, its exogenous administration may promote healing. The topical administration of SB3 is challenging due to its protein nature. Physical entrapment in wet sol-gel silica can stabilize the protein's conformation and permit its sustained delivery. However, irreversible syneresis and poor viscoelastic properties hamper wet sol-gel silica application as a semisolid vehicle. To overcome these limits, a sol-gel silica/hydroxypropylmethylcellulose (HPMC) hydrogel blend was developed. SB3 entrapped in 8% SiO2 wet sol-gel silica preserved its structure, was stabilized against denaturation, and was slowly released for at least three days. Blending a silica gel with an HPMC-glycerol (metolose-G) hydrogel permitted spreadability without affecting the protein's release kinetics. When administered in vivo, SB3 in silica/metolose-G-but not in solution or in metolose-G alone-accelerated wound healing in SB3 knockout and diabetic mouse models. The results confirmed that SB3 is a new pharmacological option for the treatment of chronic ulcers, especially when formulated in a slow-releasing vehicle. Silica-metolose-G represents a novel type of semisolid dosage form which could also be applied for the formulation of other bioactive proteins.

Impaired Hematopoietic Stem/Progenitor Cell Traffic and Multi-organ Damage in Diabetes.

During antenatal development, hematopoietic stem/progenitor cells (HSPCs) arise from a specialized endothelium and migrate from the extraembryonic mesoderm to the fetal liver before establishing hematopoiesis in the bone marrow (BM). It is still debated whether, in adulthood, HSPCs display such ontologic overlap with vascular cells and capacity for endothelial differentiation. Yet, adult HSPCs retain a prominent migratory activity and traffic in the bloodstream to secondary lymphoid organs and all peripheral tissues, before eventually returning to the BM. While patrolling parenchymatous organs, HSPCs locate close to the vasculature, where they establish local hematopoietic islands and contribute to tissue homeostasis by paracrine signals. Solid evidence shows that diabetes mellitus jeopardizes the traffic of HSPCs from BM to the circulation and peripheral tissues, a condition called "mobilopathy." A reduction in the levels of circulating HSPCs is the most immediate and apparent consequence, which has been consistently observed in human diabetes, and is strongly associated with future risk for multi-organ damage, including micro- and macro-angiopathy. But the shortage of HSPCs in the blood is only the visible tip of the iceberg. Abnormal HSPC traffic results from a complex interplay among metabolism, innate immunity, and hematopoiesis. Notably, mobilopathy is mechanistically connected with diabetes-induced myelopoiesis. Impaired traffic of HSPCs and enhanced generation of pro-inflammatory cells synergize for tissue damage and impair the resolution of inflammation. We herein summarize the current evidence that diabetes affects HSPC traffic, which are the causes and consequences of such alteration, and how it contributes to the overall disease burden.

Hematopoietic and Nonhematopoietic p66Shc Differentially Regulates Stem Cell Traffic and Vascular Response to Ischemia in Diabetes.

Aims: Peripheral artery disease (PAD) is a severe complication of diabetes, characterized by defective traffic of hematopoietic stem/progenitor cells (HSPCs). We examined the hematopoietic versus nonhematopoietic role of p66Shc in regulating HSPC traffic and blood flow recovery after ischemia in diabetic mice. Results: Using streptozotocin-induced diabetes, chimeric mice with green fluorescent protein (GFP)+ bone marrow (BM), and the hind limb ischemia model, we found that the physiologic mobilization and homing of HSPCs were abolished by diabetes, along with impaired vascular recovery. Hematopoietic deletion of p66Shc, obtained by transplanting p66Shc-/- BM cells into wild-type (Wt) recipients, but not nonhematopoietic deletion, constrained hyperglycemia-induced myelopoiesis, rescued postischemic HSPC mobilization, and improved blood flow recovery in diabetic mice. In Wt diabetic mice transplanted with BM cells from GFP+p66Shc-/- mice, the amount of HSPCs homed to ischemic muscles was greater than in mice transplanted with GFP+p66Shc+/+ cells, with recruited cells displaying higher expression of adhesion molecules and Vegf. In 40 patients with diabetes, p66Shc gene expression in mononuclear cells was correlated with myelopoiesis and elevated in the presence of PAD. In 13 patients with diabetes and PAD, p66Shc expression in HSPC-mobilized peripheral blood cells was inversely correlated with VEGF expression. Innovation: For the first time, we dissect the role of hematopoietic versus nonhematopoietic p66Shc in regulating HSPC traffic and ischemic responses. Conclusion: Hematopoietic deletion of p66Shc was sufficient to rescue HSPC mobilization and homing in diabetes after ischemia and improved blood flow recovery. Inhibiting p66Shc in blood cells may be a novel strategy to counter PAD in diabetes. Antioxid. Redox Signal. 36, 593-607. Clinical Trial No.: NCT02790957.

The BET Protein Inhibitor Apabetalone Rescues Diabetes-Induced Impairment of Angiogenic Response by Epigenetic Regulation of Thrombospondin-1.

Aims: Therapeutic modulation of blood vessel growth holds promise for the prevention of limb ischemia in diabetic (DM) patients with peripheral artery disease (PAD). Epigenetic changes, namely, posttranslational histone modifications, participate in angiogenic response suggesting that chromatin-modifying drugs could be beneficial in this setting. Apabetalone (APA), a selective inhibitor of bromodomain (BRD) and bromodomain and extraterminal containing protein family (BET) proteins, prevents bromodomain-containing protein 4 (BRD4) interactions with chromatin thus modulating transcriptional programs in different organs. We sought to investigate whether APA affects angiogenic response in diabetes. Results: Compared with vehicle, APA restored tube formation and migration in human aortic endothelial cells (HAECs) exposed to high-glucose (HG) levels. Expression profiling of angiogenesis genes showed that APA prevents HG-induced upregulation of the antiangiogenic molecule thrombospondin-1 (THBS1). ChIP-seq and chromatin immunoprecipitation (ChIP) assays in HG-treated HAECs showed the enrichment of both BRD4 and active marks (H3K27ac) on THBS1 promoter, whereas BRD4 inhibition by APA prevented chromatin accessibility and THBS1 transcription. Mechanistically, we show that THBS1 inhibits angiogenesis by suppressing vascular endothelial growth factor A (VEGFA) signaling, while APA prevents these detrimental changes. In diabetic mice with hind limb ischemia, epigenetic editing by APA restored the THBS1/VEGFA axis, thus improving limb vascularization and perfusion, compared with vehicle-treated animals. Finally, epigenetic regulation of THBS1 by BRD4/H3K27ac was also reported in DM patients with PAD compared with nondiabetic controls. Innovation: This is the first study showing that BET protein inhibition by APA restores angiogenic response in experimental diabetes. Conclusions: Our findings set the stage for preclinical studies and exploratory clinical trials testing APA in diabetic PAD. Antioxid. Redox Signal. 36, 667-684.

Fenofibrate increases circulating haematopoietic stem cells in people with diabetic retinopathy: a randomised, placebo-controlled trial.

AIM/HYPOTHESIS: In two large RCTs, fenofibrate reduced the progression of diabetic retinopathy. We investigated whether fenofibrate increases circulating haematopoietic stem/progenitor cells (HSPCs), which have vascular properties and have been shown to protect from retinopathy. METHODS: We conducted a 12 week parallel-group RCT comparing fenofibrate vs placebo. Patients with diabetic retinopathy and without other conditions that would affect HSPCs were enrolled at a tertiary diabetes outpatient clinic and randomised to receive fenofibrate or placebo based on a computer-generated sequence. Patients and study staff assessing the outcomes were blinded to group assignment. The primary endpoint was the change in the levels of circulating HSPCs, defined by expression of the stem cell markers CD34 and/or CD133. Secondary endpoints were the changes in endothelial progenitor cells, lipids, soluble mediators and gene expression. We used historical data on the association between HSPCs and retinopathy outcomes to estimate the effect of fenofibrate on retinopathy progression. RESULTS: Forty-two participants with diabetic retinopathy were randomised and 41 completed treatment and were analysed (20 in the placebo group and 21 in the fenofibrate group). Mean age was 57.4 years, diabetes duration was 18.2 years and baseline HbA1c was 60 mmol/mol (7.6%). When compared with placebo, fenofibrate significantly increased levels of HSPCs expressing CD34 and/or CD133. CD34+ HSPCs non-significantly declined in the placebo group (mean ± SD -44.2 ± 31.6 cells/106) and significantly increased in the fenofibrate group (53.8 ± 31.1 cells/106). The placebo-subtracted increase in CD34+ HSPCs from baseline was 30% (99.3 ± 43.3 cells/106; p = 0.027) which, projected onto the relationship between HSPC levels and retinopathy outcomes, yielded an OR of retinopathy progression of 0.67 for fenofibrate vs placebo. Endothelial differentiation of CD34+ cells, estimated by the %KDR (kinase insert domain receptor) expression, was significantly reduced by fenofibrate. Fenofibrate decreased serum triacylglycerols, but the change in triacylglycerols was unrelated to the change in HSPCs. No effect was observed for endothelial progenitor cells, cytokines/chemokines (stromal-cell derived factor-1, vascular endothelial growth factor, monocyte chemoattractant protein-1) and gene expression in peripheral blood mononuclear cells. CONCLUSIONS/INTERPRETATION: Fenofibrate increased HSPC levels in participants with diabetic retinopathy and this mechanism may explain why fenofibrate reduced retinopathy progression in previous studies. TRIAL REGISTRATION: ClinicalTrials.gov NCT01927315.

Estrogen Receptor Functions and Pathways at the Vascular Immune Interface.

Estrogen receptor (ER) activity mediates multiple physiological processes in the cardiovascular system. ERα and ERß are ligand-activated transcription factors of the nuclear hormone receptor superfamily, while the G protein-coupled estrogen receptor (GPER) mediates estrogenic signals by modulating non-nuclear second messengers, including activation of the MAP kinase signaling cascade. Membrane localizations of ERs are generally associated with rapid, non-genomic effects while nuclear localizations are associated with nuclear activities/transcriptional modulation of target genes. Gender dependence of endothelial biology, either through the action of sex hormones or sex chromosome-related factors, is becoming increasingly evident. Accordingly, cardiometabolic risk increases as women transition to menopause. Estrogen pathways control angiogenesis progression through complex mechanisms. The classic ERs have been acknowledged to function in mediating estrogen effects on glucose metabolism, but 17ß-estradiol also rapidly promotes endothelial glycolysis by increasing glucose transporter 1 (GLUT1) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) levels through GPER-dependent mechanisms. Estrogens alter monocyte and macrophage phenotype(s), and induce effects on other estrogen-responsive cell lineages (e.g., secretion of cytokines/chemokines/growth factors) that impact macrophage function. The pharmacological modulation of ERs for therapeutic purposes, however, is particularly challenging due to the lack of ER subtype selectivity of currently used agents. Identifying the determinants of biological responses to estrogenic agents at the vascular immune interface and developing targeted pharmacological interventions may result in novel improved therapeutic solutions.

Arrhythmogenic Cardiomyopathy Is a Multicellular Disease Affecting Cardiac and Bone Marrow Mesenchymal Stromal Cells.

Arrhythmogenic cardiomyopathy (AC) is a familial cardiac disorder at high risk of arrhythmic sudden death in the young and athletes. AC is hallmarked by myocardial replacement with fibro-fatty tissue, favoring life-threatening cardiac arrhythmias and contractile dysfunction. The AC pathogenesis is unclear, and the disease urgently needs mechanism-driven therapies. Current AC research is mainly focused on 'desmosome-carrying' cardiomyocytes, but desmosomal proteins are also expressed by non-myocyte cells, which also harbor AC variants, including mesenchymal stromal cells (MSCs). Consistently, cardiac-MSCs contribute to adipose tissue in human AC hearts. We thus approached AC as a multicellular disorder, hypothesizing that it also affects extra-cardiac bone marrow (BM)-MSCs. Our results show changes in the desmosomal protein profile of both cardiac- and BM- MSCs, from desmoglein-2 (Dsg2)-mutant mice, accompanied with profound alterations in cytoskeletal organization, which are directly caused by AC-linked DSG2 downregulation. In addition, AC BM-MSCs display increased proliferation rate, both in vitro and in vivo, and, by using the principle of the competition homing assay, we demonstrated that mutant circulating BM-MSCs have increased propensity to migrate to the AC heart. Taken altogether, our results indicate that cardiac- and BM- MSCs are additional cell types affected in Dsg2-linked AC, warranting the novel classification of AC as a multicellular and multiorgan disease.

Inhibition of SGLT2 Rescues Bone Marrow Cell Traffic for Vascular Repair: Role of Glucose Control and Ketogenesis.

The mechanisms by which sodium-glucose cotransporter 2 inhibitors (SGLT2i) improve cardiovascular outcomes in people with diabetes are incompletely understood. Recent studies show that SGLT2i may increase the levels of circulating cells with vascular regenerative capacity, at least in part by lowering glycemia. In this study, we used mice with streptozotocin-induced diabetes treated with the SGLT2i dapagliflozin at a dose that reduced glucose levels by 20%. Dapagliflozin improved the diabetes-associated defect of hematopoietic stem cell mobilization after stimulation with granulocyte colony-stimulating factor. Dapagliflozin rescued the traffic of bone marrow (BM)-derived cells to injured carotid arteries and improved endothelial healing in diabetic mice. Defective homing of CD49d+ granulocytes was causally linked with impaired endothelial repair and was reversed by dapagliflozin. The effects of dapagliflozin were mimicked by a similar extent of glucose reduction achieved with insulin therapy and by a ketone drink that artificially elevated ß-hydroxybutyrate. Inhibition of endothelial repair by resident cells using the CXCR4 antagonist AMD3100 did not abolish the vascular effect of dapagliflozin, indirectly supporting that endothelial healing by dapagliflozin was mediated by recruitment of circulating cells. In summary, we show that dapagliflozin improved the traffic of BM-derived hematopoietic cells to the site of vascular injury, providing a hitherto unappreciated mechanism of vascular protection.

Diabetes pharmacotherapy and circulating stem/progenitor cells. State of the art and evidence gaps.

Diabetes is burdened with the development of several end-organ complications leading to excess mortality. Though the causes of such organ damage are far from being clarified, diabetes has been redefined as a disease of impaired damage control, wherein ongoing damage is not adequately compensated by activation of repair processes. Bone marrow-derived hematopoietic stem/progenitor cells (HSPCs) and their descendants endothelial progenitor cells (EPCs) have been extensively studied as major players in tissue homeostasis as well as biomarkers of diabetic complication risk. Thus, strategies to raise the levels of circulating HSPCs/EPCs have attracted interest for their potential to modify the future risk of complications. We herein discuss state-of-the-art of the effects exerted by diabetes pharmacotherapy on such cell populations. Further, we highlight which outstanding questions remain to be addressed for a more comprehensive understanding of this topic.

Non-genomic mechanisms in the estrogen regulation of glycolytic protein levels in endothelial cells.

Few studies have explored the mechanisms coupling estrogen signals to metabolic demand in endothelial cells. We recently showed that 17ß-estradiol (E2) triggers angiogenesis via the membrane G-protein coupled estrogen receptor (GPER) and the key glycolytic protein PFKFB3 as a downstream effector. We herein investigated whether estrogenic agents regulate the stability and/or degradation of glycolytic proteins in human umbilical vein endothelial cells (HUVECs). Similarly to E2, the GPER selective agonist G1 rapidly increased PFKFB3 protein amounts, without affecting mRNA levels. In the presence of cycloheximide, E2 and G1 treatment counteracted PFKFB3 degradation over time, whereas E2-induced PFKFB3 stabilization was abolished by the GPER antagonist G15. Inhibitors of selective SCF E3 ubiquitin ligase (SMER-3) and proteasome (MG132) rapidly increased PFKFB3 protein levels. Accordingly, ubiquitin-bound PFKFB3 was lower in E2- or G1-treated HUVECs. Both agents increased deubiquitinase USP19 levels through GPER signaling. Notably, USP 19 siRNA decreased PFKFB3 levels and abolished E2- and G1-mediated HUVEC tubularization. Finally, E2 and G1 treatments rapidly enhanced glucose transporter GLUT1 levels via GPER independent of transcriptional activation. These findings provide new evidence on mechanisms coupling estrogen signals with the glycolytic program in endothelium and unravel the role of USP19 as a target of the pro-angiogenic effect of estrogenic agents.

Pharmacologic PPAR-γ Activation Reprograms Bone Marrow Macrophages and Partially Rescues HSPC Mobilization in Human and Murine Diabetes.

Mobilization of hematopoietic stem/progenitor cells (HSPC) from the bone marrow (BM) is impaired in diabetes. Excess oncostatin M (OSM) produced by M1 macrophages in the diabetic BM signals through p66Shc to induce Cxcl12 in stromal cells and retain HSPC. BM adipocytes are another source of CXCL12 that blunts mobilization. We tested a strategy of pharmacologic macrophage reprogramming to rescue HSPC mobilization. In vitro, PPAR-γ activation with pioglitazone switched macrophages from M1 to M2, reduced Osm expression, and prevented transcellular induction of Cxcl12 In diabetic mice, pioglitazone treatment downregulated Osm, p66Shc, and Cxcl12 in the hematopoietic BM, restored the effects of granulocyte-colony stimulation factor (G-CSF), and partially rescued HSPC mobilization, but it increased BM adipocytes. Osm deletion recapitulated the effects of pioglitazone on adipogenesis, which was p66Shc independent, and double knockout of Osm and p66Shc completely rescued HSPC mobilization. In the absence of OSM, BM adipocytes produced less CXCL12, being arguably devoid of HSPC-retaining activity, whereas pioglitazone failed to downregulate Cxcl12 in BM adipocytes. In patients with diabetes on pioglitazone therapy, HSPC mobilization after G-CSF was partially rescued. In summary, pioglitazone reprogrammed BM macrophages and suppressed OSM signaling, but sustained Cxcl12 expression by BM adipocytes could limit full recovery of HSPC mobilization.

Diabetes mellitus impairs circulating proangiogenic granulocytes.

AIMS/HYPOTHESIS: Cardiovascular risk in diabetes is at least in part attributable to defective angiogenesis. Since diabetes negatively affects blood cells involved in angiogenesis, we herein evaluated whether diabetes impairs proangiogenic granulocytes (PAGs). METHODS: We characterised and quantified PAGs as CD49d+ granulocytes in peripheral blood of participants with type 2 or type 1 diabetes and in non-diabetic control participants. We evaluated PAG antigenic profile and assessed in vitro functional properties of CD49d+ granulocytes using 2D and 3D angiogenesis assays. We also quantified PAGs before and after glucose control with a sodium-glucose cotransporter 2 (SGLT2) inhibitor, dapagliflozin. In parallel, we measured Ly6G+CD49d+ PAGs in streptozotocin-induced type 1-like diabetic mice vs non-diabetic control mice. RESULTS: PAGs were composed of eosinophils (>80%) and neutrophils (<20%). Within both populations, CD49d identified CXCR4high/VEGFR1high cells. CD49d+ granulocytes supported in vitro angiogenesis by endothelial cells significantly more than CD49d- control granulocytes, and physically interacted with endothelial cells. Granulocytes from type 2 diabetic participants had a profoundly impaired capacity to stimulate endothelial cell tubule formation compared with those from non-diabetic control participants. CD49d+ PAGs were reduced by 30-40% and were functionally impaired in diabetic vs control individuals. PAG levels inversely correlated with plasma glucose (r = -0.25; p = 0.025) and significantly increased 1.8-times after glucose control with dapagliflozin, which reduced HbA1c by 1.0% (11 mmol/mol). Levels of Ly6G+CD49d+ PAGs were also significantly reduced also in type 1 diabetic mice vs control mice. CONCLUSIONS/INTERPRETATION: We illustrate a significant impairment of PAGs in diabetes and provide evidence for a direct role of hyperglycaemia. These findings add mechanistic information to explain the defective angiogenesis in diabetes. Graphical abstract.