Roberta Foresti

People living with HIV (PWH) who take integrase strand transfer inhibitors (INSTIs), such as dolutegravir, may gain weight and fat, which increases the risk of cardiometabolic diseases. We previously showed that INSTIs induce adipocyte hypertrophy, insulin resistance, fibrosis, and mitochondrial dysfunction. Since INSTIs are currently the primary antiretroviral treatment used to control HIV infection in PWH, it is crucial to identify strategies that mitigate their adverse effects on weight and adipose tissue function. Glucagon-like peptide-1 (GLP-1) receptor agonists, such as liraglutide, and dual GLP-1/glucose-dependent insulinotropic polypeptide (GIP) receptor agonists (RAs), such as tirzepatide, promote weight loss even in those PWH treated with an INSTI. Here, we evaluated whether these molecules could directly mitigate the deleterious effects of dolutegravir in human adipocytes differentiated from adipose stromal cells. Liraglutide and tirzepatide had no or minimal effects on control adipocytes. As expected, dolutegravir induced lipid accumulation and lipogenesis, reduced lipolysis, and induced fibrosis, oxidative stress, mitochondrial dysfunction, and insulin resistance. Liraglutide and tirzepatide counteracted all the effects of dolutegravir except for mitochondrial dysfunction. Using LAGIPRA, a selective GIP-RA, and exendin 9-39, a GLP-1R selective antagonist, we revealed that the ability of liraglutide and tirzepatide to reverse the adverse effects of dolutegravir mainly involves GLP-1R activation. Altogether, our results showed that GLP-1 and dual GLP-1/GIP RAs can alleviate INSTI-induced adipocyte dysfunction and insulin resistance. GLP-1 and GLP-1/GIP RAs represent an effective strategy for mitigating INSTI-induced weight gain and insulin resistance, which would likely result in an improved cardiometabolic profile in PWH.

Heme is an iron-containing tetrapyrrole with dual biological functions. While it serves as an essential prosthetic group in various hemoproteins, heme is cytotoxic in its ‘free’, non-protein-bound, form. Labile heme (LH) denotes the intracellular fraction of bioavailable heme that is readily exchangeable for incorporation into hemoproteins. To investigate the regulatory role of this heme fraction in inflammatory activated macrophages, we applied the selective fluorescent small molecule H-FluNox for LH detection in lipopolysaccharide (LPS)-stimulated murine bone marrow-derived macrophages (BMDMs). Studies with H-FluNox and its cell-permeable derivative acetylated (Ac)-H-FluNox revealed a time-dependent decrease of LH levels in living BMDMs upon treatment with LPS. Expression of d-aminolevulinate synthase 1 (ALAS1), the rate-limiting enzyme of heme synthesis, was up-regulated in parallel to the decrease of LH levels. Studies in subcellular organelles of BMDMs demonstrated that LH concentrations were markedly higher in mitochondria compared to cytosol and nuclei. Furthermore, expression of inducible nitric oxide synthase (iNOS), a heme-containing pro-inflammatory enzyme, was dependent on intracellular LH concentrations. Specifically, LPS-dependent iNOS induction was attenuated in BMDMs displaying decreased LH, either after treatment with pharmacological heme synthesis inhibitors, or with genetic deficiency of the nuclear heme sensor BACH1. By contrast, inducibility of iNOS by LPS was markedly higher in BMDMs exhibiting increased levels of LH following treatment with the heme synthesis substrate d-aminolevulinate (ALA). Finally, pharmacological inhibition of succinate dehydrogenase, which enhances intracellular levels of ALA and LH, also caused higher inducibility of iNOS by LPS. In conclusion, the data indicate that intracellular LH is modulated by inflammatory stimulation in mouse macrophages and is critical for heme incorporation into the iNOS hemoprotein. Thus, heme availability may represent a regulatory link between metabolic and inflammatory pathways.

Carbon monoxide (CO), a gas endogenously produced in mammalian tissues, exerts vasodilatory, anti‐ischemic, and anti‐inflammatory effects. These properties have prompted the development of CO‐releasing molecules (CO‐RMs) for therapeutic purposes. Among this class of compounds is CORM‐A1, a boron‐based carboxylic acid, which generates controlled amounts of CO under physiological conditions. In this proof‐of‐principle study we explored the potential of CORM‐A1 to protect kidneys from warm ischemia and reperfusion (WI/R) injury in rat and swine models. We found that intravenous administration of CORM‐A1 significantly increased blood carboxyhemoglobin (COHb) levels while facilitating CO accumulation in renal tissue, thus confirming its ability to deliver CO to peripheral organs. In rats subjected to 45‐ and 60‐min WI/R, administration of CORM‐A1 improved renal function at reperfusion, as shown by decreased serum creatinine and urea levels. Histopathological analysis revealed substantial protection against tubular damage, cell infiltration, and inflammation, especially after 60‐min ischemia. Protection was dose‐dependent, with higher doses offering enhanced effects. In a swine kidney auto‐transplantation model, CORM‐A1 significantly improved graft function, reduced fibrosis and necrosis, and extended graft survival. These findings position CORM‐A1 as a promising CO prodrug, with translational relevance for clinical applications in kidney transplantation and other ischemia‐related conditions.

Origanum majorana L. (O. majorana) (Lamiaceae) is an aromatic Mediterranean plant widely used in food, cosmetics, and traditional medicine due to its aroma and rich content of bioactive compounds. While its leaves and flowers are commonly utilized, lignified stems are often discarded. This study compared hydroalcoholic extracts from the leaves and flowers (valuable fraction, VF) and stems (by-product, BP). Phytochemical analysis revealed qualitatively similar profiles, identifying 20 phenolic compounds, with Rosmarinic acid and Salvianolic acid B as the most and second most abundant, respectively. Antioxidant activity was evaluated in vitro using DPPH (IC₅₀ [µg/mL]: VF 30.11 ± 3.46; BP 31.72 ± 1.46), H₂O₂ (IC₅₀ [µg/mL]: VF 103.09 ± 4.97; BP 119.55 ± 10.58), and O₂^•− (IC₅₀ [µg/mL]: VF 0.71 ± 0.062; BP 0.79 ± 0.070). Both extracts (20 µg/mL) fully restored oxidative balance in hemin-stressed AC16 cardiomyocytes, without altering the expression of catalase, heme-oxygenase 1, superoxide dismutase 2, or ferritin. Anti-inflammatory activity in LPS-stimulated RAW 264.7 macrophages showed that VF (IC₅₀ 400 µg/mL) reduced ^•NO release to control levels, while BP achieved a ~60% reduction. Cytotoxicity was assessed on cancer cell lines: CaCo-2 (IC₅₀ [µg/mL]: VF 154.1 ± 6.22; BP 305.2 ± 15.94), MCF-7 (IC₅₀ [µg/mL]: VF 624.6 ± 10.27; BP 917.9 ± 9.87), and A549 (IC₅₀ [µg/mL]: VF 720.8 ± 13.66; BP 920.2 ± 16.79), with no cytotoxicity on normal fibroblasts HFF-1 (IC₅₀ > 1000 µg/mL for both extracts). Finally, both extracts slightly inhibited only CYP1A2 (IC₅₀ [µg/mL]: VF 497.45 ± 9.64; BP 719.72 ± 11.37) and CYP2D6 (IC₅₀ [µg/mL]: VF 637.15 ± 14.78, BP 588.70 ± 11.01). These results support the potential reuse of O. majorana stems as a sustainable source of bioactive compounds for nutraceutical and health-related applications.

Metabolic reprograming has been linked to epithelial-to-mesenchymal transition (EMT) in cancer cells, but how it influences EMT in normal cells remains largely unknown. Here we explored how metabolism impacts delamination and migration of avian trunk neural crest cells, an important progenitor cell population of the vertebrate embryo. We report that delamination exhibits a quiescent metabolic phenotype whereas migration is characterized by OXPHOS-driven metabolism coupled to distinct expression of metabolic, EMT and developmental genes. While glucose and glutamine are required for delamination and migration, we uncover a specific role for glutamine and its catabolizing enzyme glutaminase in the unfolding of NCC delamination. Namely, glutamine is required for nuclear accumulation of glutaminase, which interacts and cooperates with Wnt signaling to regulate EMT gene expression and cell cycle during delamination. Our data indicate that similarly to cancer cells, embryonic cells engage metabolic enzymes for non-canonical signaling functions to connect metabolism with EMT.

Diabetic retinopathy (DR) is a microvascular complication of diabetes mellitus and a leading cause of blindness in the working-age population. Current pharmacological treatments counteract DR’s later stages without targeting the earlier disease phases. Using computational approaches, our group previously identified the α1D and α2C adrenoceptors (α1DR and α2CR) as new putative drug targets for DR. Therefore, the aim of this work was to validate the role of these receptors in an in vitro model of DR, i.e., retinal pigmented epithelial cells (ARPE-19) challenged with high glucose (HG, 50 mM). We examined the effects of selective α1DR and α2CR agonists and antagonists on hyperglycemia-induced mitochondrial dysfunction and blood retinal barrier breakdown. Seahorse XFe was employed to assess the oxygen consumption rate and extracellular acidification rate. The integrity of the ARPE-19 barrier was evaluated through transepithelial electrical resistance measurements and a sodium fluorescein permeability test. α1DR pharmacological modulation through the α1DR antagonist BMY 7378 (0.1–1 µM, 24 h), but not α2CR, significantly attenuated HG-induced mitochondrial dysfunction. BMY 7378 (0.1–1 µM, 48 h) also prevented HG-mediated damage to retinal epithelial integrity. In contrast, the α1DR agonist phenylephrine (1–10 μM, 24 h) further reduced ARPE-19 mitochondrial activity compared to HG, indicating that α1D activation is directly implicated in DR-mediated mitochondrial dysfunction. In conclusion, the current in vitro study validated α1DR as a pharmacological target for DR.

Acute hyperhemolysis is a severe life-threatening complication in patients with sickle cell disease (SCD) that may occur during delayed hemolytic transfusion reaction (DHTR), or vaso-occlusive crises associated with multiorgan failure. Here, we developed in vitro and in vivo animal models to mimic endothelial damage during the early phase of hyperhemolysis in SCD. We then used the carbon monoxide (CO)–releasing molecule CORM-401 and examined its effects against endothelial activation, damage, and inflammation inflicted by hemolysates containing red blood cell membrane–derived particles. The in vitro results revealed that CORM-401: (1) prevented the upregulation of relevant proinflammatory and proadhesion markers controlled by the NF-κB enhancer of activated B cells, and (2) abolished the expression of the nuclear factor erythroid-2-related factor 2 (Nrf2) that regulates the inducible antioxidant cell machinery. We also show in SCD mice that CORM-401 protects against hemolysate-induced acute damage of target organs such as the lung, liver, and kidney through modulation of NF-κB proinflammatory and Nrf2 antioxidant pathways. Our data demonstrate the efficacy of CORM-401 as a novel therapeutic agent to counteract hemolysate-induced organ damage during hyperhemolysis in SCD. This approach might be considered as possible preventive treatment in high-risk situations such as patients with SCD with history of DHTR.

Metabolic reprograming has been linked to epithelial-to-mesenchymal transition (EMT) in cancer cells, but how it influences EMT in normal cells remains largely unknown. Here we explored how metabolism impacts delamination and migration of avian trunk neural crest cells, an important stem cell population of the vertebrate embryo. We report that delamination exhibits a quiescent metabolic phenotype whereas migration is characterized by OXPHOS-driven metabolism coupled to distinct expression of metabolic, EMT and developmental genes. While glucose and glutamine are required for delamination and migration, we uncover a novel role for glutamine and its catabolizing enzyme glutaminase in delamination. Specifically, glutamine is required for nuclear translocation of glutaminase, which interacts and cooperates with Wnt signaling to regulate EMT gene expression and cell cycle during delamination. Our data indicate that similarly to cancer cells, embryonic cells engage metabolic enzymes for non-canonical signaling functions to connect metabolism with EMT.