Synthesis and in vitro cytotoxic activity of dye-linker-macrocycle conjugates with variable linker length and components.

The study investigated the structure-activity relationship of newly synthesized dye-linker-macrocycle (DLM) conjugates and the effect of each component on various biological properties, including cytotoxicity, cellular uptake, intracellular localization, interaction with DNA and photodynamic effects. The conjugates were synthesized by combining 1,8-naphthalimide and thioxanthone dyes with 1,4,7,10-tetraazacyclododecane (cyclen) and 1-aza-12-crown-4 (1A12C4) using alkyl linkers of different lengths. The results revealed significant differences in biological activity among the various series of conjugates. Particularly, 1A12C4 conjugates exhibited notably higher cytotoxicity compared to cyclen conjugates. Conjugation with 1A12C4 proved to be an effective strategy for increasing cellular uptake and cytotoxicity of small-molecule conjugates. In addition, the results highlighted the critical role of linker length in modulating the biological activity of DLM conjugates. It became clear that the choice of each component (dye, macrocycle and linker) could significantly alter the biological activity of the conjugates.

Hybrid, dual visible and near-infrared fluorescence emission of (6,5) single-walled carbon nanotubes modified with fluorescein through aryl diazonium salt chemistry.

The aryl diazonium salt chemistry offers enhancement of near-infrared (NIR) emission of single-walled carbon nanotubes (SWCNTs), although, the attachment of functional molecules which could bring hybrid properties through the process is underdeveloped. In this work, we utilize aryl diazonium salt of fluorescein to createsp3defects on (6,5) SWCNTs. We study the influence of pH on the grafting process identifying that pH 5-6 is necessary for a successful reaction. The fluorescein-modified (6,5) SWCNTs (F-(6,5) SWCNTs) exhibit red-shiftedE11* emission in the NIR region attributed to luminescentsp3defects, but also visible (Vis) fluorescence at 515 nm from surface-attached fluorescein molecules. The fluorescence in both Vis and NIR regions of F-(6,5) SWCNTs exhibit strong pH-dependency associated with the dissociation of fluorescein molecules with an indication of photoinduced-electron transfer quenching the Vis emission of fluorescein dianion. The F-(6,5) SWCNTs could potentially be used for dual-channel medical imaging as indicated by our preliminary experiments. We hope that our research will encourage new, bold modifications of SWCNTs with functional molecules introducing new, unique hybrid properties.

Surprising Solid-State ESIPT Emission from Apparently Ordinary Salicyliden Glycinates Schiff Bases.

Excited-State Intramolecular Photon Transfer (ESIPT) is known for the geometry-related phenolic and imine groups. The Schiff bases formed upon condensation of salicyl aldehyde and glycine led to the formation of ESIPT models. A series of alkali metal salicyliden glycinates were analyzed by X-ray diffraction of their monocrystals and spectroscopy measurements. The X-ray analysis revealed varied hydration levels between the salts. They adapted trans geometry on the imine groups and mostly anticlinal conformation with the neighboring atoms, which is different from the other structurally-related compounds in literature. Fluorescence of these compounds was found for the crystalline forms only. Protonation of the imine nitrogen atom and further proton distribution was consistent with the ESIPT theory, which also explained the observed fluorescence with the highest Stokes shift of 10,181 cm-1 and 10.1% of fluorescence quantum yield for the sodium salt.

Hexokinase 2 Inhibition and Biological Effects of BNBZ and Its Derivatives: The Influence of the Number and Arrangement of Hydroxyl Groups.

Hexokinase 2 (HK2), an enzyme of the sugar kinase family, plays a dual role in glucose metabolism and mediating cancer cell apoptosis, making it an attractive target for cancer therapy. While positive HK2 expression usually promotes cancer cells survival, silencing or inhibiting this enzyme has been found to improve the effectiveness of anti-cancer drugs and even result in cancer cell death. Previously, benitrobenrazide (BNBZ) was characterized as a potent HK2 inhibitor with good anti-cancer activity in mice, but the effect of its trihydroxy moiety (pyrogallol-like) on inhibitory activity and some cellular functions has not been fully understood. Therefore, the main goal of this study was to obtain the parent BNBZ (2a) and its three dihydroxy derivatives 2b-2d and to conduct additional physicochemical and biological investigations. The research hypothesis assumed that the HK2 inhibitory activity of the tested compounds depends on the number and location of hydroxyl groups in their chemical structure. Among many studies, the binding affinity to HK2 was determined and two human liver cancer cell lines, HepG2 and HUH7, were used and exposed to chemicals at various times: 24 h, 48 h and 72 h. The study showed that the modifications to the structures of the new BNBZ derivatives led to significant changes in their activities. It was also found that these compounds tend to aggregate and exhibit toxic effects. They were found to contribute to: (a) DNA damage, (b) increased ROS production, and (c) disruption of cell cycle progression. It was observed that, HepG2, occurred much more sensitive to the tested chemicals than the HUH7 cells; However, regardless of the used cell line it seems that the increase in the expression of HK2 in cancer cells compared to normal cells which have HK2 at a very low level, is a serious obstacle in anti-cancer therapy and efforts to find the effective inhibitors of this enzyme should be intensified.

Nonnutritive sweetener consumption during pregnancy, adiposity, and adipocyte differentiation in offspring: evidence from humans, mice, and cells.

BACKGROUND: Obesity often originates in early life, and is linked to excess sugar intake. Nonnutritive sweeteners (NNS) are widely consumed as "healthier" alternatives to sugar, yet recent evidence suggests NNS may adversely influence weight gain and metabolic health. The impact of NNS during critical periods of early development has rarely been studied. We investigated the effect of prenatal NNS exposure on postnatal adiposity and adipocyte development. METHODS: In the CHILD birth cohort (N = 2298), we assessed maternal NNS beverage intake during pregnancy and child body composition at 3 years, controlling for maternal BMI and other potential confounders. To investigate causal mechanisms, we fed NNS to pregnant C57BL6J mice at doses relevant to human consumption (42 mg/kg/day aspartame or 6.3 mg/kg/day sucralose), and assessed offspring until 12 weeks of age for: body weight, adiposity, adipose tissue morphology and gene expression, glucose and insulin tolerance. We also studied the effect of sucralose on lipid accumulation and gene expression in cultured 3T3-L1 pre-adipocyte cells. RESULTS: In the CHILD cohort, children born to mothers who regularly consumed NNS beverages had elevated body mass index (mean z-score difference +0.23, 95% CI 0.05-0.42 for daily vs. no consumption, adjusted for maternal BMI). In mice, maternal NNS caused elevated body weight, adiposity, and insulin resistance in offspring, especially in males (e.g., 47% and 15% increase in body fat for aspartame and sucralose vs. controls, p < 0.001). In cultured adipocytes, sucralose exposure at early stages of differentiation caused increased lipid accumulation and expression of adipocyte differentiation genes (e.g., C/EBP-α, FABP4, and FASN). These genes were also upregulated in adipose tissue of male mouse offspring born to sucralose-fed dams. CONCLUSION: By triangulating evidence from humans, mice, and cultured adipocytes, this study provides new evidence that maternal NNS consumption during pregnancy may program obesity risk in offspring through effects on adiposity and adipocyte differentiation.

Exacerbation of Neonatal Hemolysis and Impaired Renal Iron Handling in Heme Oxygenase 1-Deficient Mice.

In most mammals, neonatal intravascular hemolysis is a benign and moderate disorder that usually does not lead to anemia. During the neonatal period, kidneys play a key role in detoxification and recirculation of iron species released from red blood cells (RBC) and filtered out by glomeruli to the primary urine. Activity of heme oxygenase 1 (HO1), a heme-degrading enzyme localized in epithelial cells of proximal tubules, seems to be of critical importance for both processes. We show that, in HO1 knockout mouse newborns, hemolysis was prolonged despite a transient state and exacerbated, which led to temporal deterioration of RBC status. In neonates lacking HO1, functioning of renal molecular machinery responsible for iron reabsorption from the primary urine (megalin/cubilin complex) and its transfer to the blood (ferroportin) was either shifted in time or impaired, respectively. Those abnormalities resulted in iron loss from the body (excreted in urine) and in iron retention in the renal epithelium. We postulate that, as a consequence of these abnormalities, a tight systemic iron balance of HO1 knockout neonates may be temporarily affected.

Unprecedented Water Effect as a Key Element in Salicyl-Glycine Schiff Base Synthesis.

Salens, as chelating, double Schiff base ligands, are an important group utilized in transition metal catalysis. They have been used to build interesting functional metal-organic frameworks (MOFs). However, salens interacting with amino acids have also found applications in receptors. Here, we intended to form a "green" glycine-derived salen fragment, but the available literature data were contradictory. Therefore, we optimized the synthetic conditions and obtained the desired product as two different crystallographic polymorphs (orthorhombic Pcca and monoclinic P21/c space groups). Their structures differ in conformation at the glycine moiety, and the monoclinic form contains additional, disordered water molecules. Despite the high stability of Schiff bases, these newly obtained compounds hydrolyze in aqueous media, the process being accelerated by metal cations. These studies, accompanied by mechanistic considerations and solid-state moisture and thermal analysis, clarify the structure and behavior of this amino acid Schiff base and shed new light on the role of water in its stability.

Synthesis of amide and sulfonamide substituted N-aryl 6-aminoquinoxalines as PFKFB3 inhibitors with improved physicochemical properties.

In oncology, the "Warburg effect" describes the elevated production of energy by glycolysis in cancer cells. The ubiquitous and hypoxia-induced 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) plays a noteworthy role in the regulation of glycolysis by producing fructose-2,6-biphosphate (F-2,6-BP), a potent activator of the glycolysis rate-limiting phosphofructokinase PFK-1. Series of amides and sulfonamides derivatives based on a N-aryl 6-aminoquinoxaline scaffold were synthesized and tested for their inhibition of PFKFB3 in vitro in a biochemical assay as well as in HCT116 cells. The carboxamide series displayed satisfactory kinetic solubility and metabolic stability, and within this class, potent lead compounds with low nanomolar activity have been identified with a suitable profile for further in vivo evaluation.

Splenic Ly6Chi monocytes contribute to adverse late post-ischemic left ventricular remodeling in heme oxygenase-1 deficient mice.

Heme oxygenase-1 (Hmox1) is a stress-inducible protein crucial in heme catabolism. The end products of its enzymatic activity possess anti-oxidative, anti-apoptotic and anti-inflammatory properties. Cardioprotective effects of Hmox1 were demonstrated in experimental models of myocardial infarction (MI). Nevertheless, its importance in timely resolution of post-ischemic inflammation remains incompletely understood. The aim of this study was to determine the role of Hmox1 in the monocyte/macrophage-mediated cardiac remodeling in a mouse model of MI. Hmox1 knockout (Hmox1-/-) and wild-type (WT, Hmox1+/+) mice were subjected to a permanent ligation of the left anterior descending coronary artery. Significantly lower incidence of left ventricle (LV) free wall rupture was noted between 3rd and 5th day after MI in Hmox1-/- mice resulting in their better overall survival. Then, starting from 7th until 21st day post-MI a more potent deterioration of LV function was observed in Hmox1-/- than in the surviving Hmox1+/+ mice. This was accompanied by higher numbers of Ly6Chi monocytes in peripheral blood, as well as higher expression of monocyte chemoattractant protein-1 and adhesion molecules in the hearts of MI-operated Hmox1-/- mice. Consequently, a greater post-MI monocyte-derived myocardial macrophage infiltration was noted in Hmox1-deficient individuals. Splenectomy decreased the numbers of circulating inflammatory Ly6Chi monocytes in blood, reduced the numbers of proinflammatory cardiac macrophages and significantly improved the post-MI LV function in Hmox1-/- mice. In conclusion, Hmox1 deficiency has divergent consequences in MI. On the one hand, it improves early post-MI survival by decreasing the occurrence of cardiac rupture. Afterwards, however, the hearts of Hmox1-deficient mice undergo adverse late LV remodeling due to overactive and prolonged post-ischemic inflammatory response. We identified spleen as an important source of these cardiovascular complications in Hmox1-/- mice.

Evaluation of biological properties of 3,3',4,4'-benzophenonetetracarboxylic dianhydride derivatives and their ability to inhibit hexokinase activity.

This investigation has explored the properties of 3,3',4,4'-benzophenonetetracarboxylic dianhydride (BDTA) derivatives with regard to their being prospective inhibitors of hexokinase II (HKII). A pluripotent embryonic carcinoma cell line P19 (ECC), was used as the biological target for newly generated potential inhibitors of HKII. The results obtained from Virtual High-Throughput Screening (VHTS), molecular modeling and biological activity studies showed BDTA to be a promising leading structure with a good binding score and simplest functionalization. The inhibitory effect was measured after 72h incubation. Of selected BDTA derivatives, the most active was compound 3b, containing 3-hydroxyphenyl moiety in the para position, being able at 100µM to decrease the mass of differentiated P19dCs cells by 30%, changing both the mitochondrial transmembrane potential and reactive oxygen species level. Under these conditions, only compound 3b had the ability to decrease hexokinase activity in a dose-dependent manner.

Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection.

Purpose: The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the lingering threat to public health has fueled the search for effective therapeutics to treat SARS-CoV-2. This study aimed to develop lipid nanoparticle (LNP) inhibitors of SARS-CoV-2 entry to reduce viral infection in the nose and upper airway. Methods: Two types of LNP formulations were prepared following a microfluidic mixing method. The LNP-Trap consisted of DOPC, DSPC, cholesterol, and DSPE-PEG-COOH modified with various spike protein binding ligands, including ACE2 peptide, recombinant human ACE2 (rhACE2) or monoclonal antibody to spike protein (mAb). The LNP-Trim consisted of ionizing cationic DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG lipids encapsulating siACE2 or siTMPRSS2. Both formulations were assayed for biocompatibility and cell uptake in airway epithelial cells (Calu-3). Functional assessment of activity was performed using SARS-CoV-2 spike protein binding assays (LNP-Trap), host receptor knockdown (LNP-Trim), and SARS-CoV-2 pseudovirus neutralization assay (LNP-Trap and LNP-Trim). Localization and tissue distribution of fluorescently labeled LNP formulations were assessed in mice following intranasal administration. Results: Both LNP formulations were biocompatible based on cell impedance and MTT cytotoxicity studies in Calu-3 cells at concentrations as high as 1 mg/mL. LNP-Trap formulations were able to bind spike protein and inhibit pseudovirus infection by 90% in Calu-3 cells. LNP-Trim formulations reduced ACE2 and TMPRSS2 at the mRNA (70% reduction) and protein level (50% reduction). The suppression of host targets in Calu-3 cells treated with LNP-Trim resulted in over 90% inhibition of pseudovirus infection. In vivo studies demonstrated substantial retention of LNP-Trap and LNP-Trim in the nasal cavity following nasal administration with minimal systemic exposure. Conclusion: Both LNP-Trap and LNP-Trim formulations were able to safely and effectively inhibit SARS-CoV-2 pseudoviral infection in airway epithelial cells. These studies provide proof-of-principle for a localized treatment approach for SARS-CoV-2 in the upper airway.

[Endothelium in physiology and pathogenesis of diseases]. / Sródblonek w fizjologii i patogenezie chorób.

Vascular endothelium is the layer of cells that line blood vessels and serve as the primary barrier between the blood and the tissues. These cells play many important functions such as regulation of the vascular tone, blood flow and pressure control, maintaining the balance between thrombosis and fibrinolysis, participation in immune system reactions and new blood vessels formation. Disturbance in any of these functions may contribute to the development of different diseases such as for e.g. hypertension, atherosclerosis and deep vein thrombosis, as well as cancer.

S/Se-Terchalcogenophene-C60 Dyads: Synthesis and Characterization of Optical and Photosensitizing Properties.

Fullerenes have been long investigated for application as singlet oxygen sources. Even though they possess high photosensitizing efficiency, their practical use is still limited, mostly because of insufficient absorption of visible and/or near-infrared light. This limitation can be overcome by introducing organic chromophores that absorb longer-wavelength light, either by covalent attachment to C60 or by its encapsulation in a polymeric matrix. In this work, we investigated the photosensitizing properties of the C60 molecule functionalized with organic units comprising thiophene or selenophene rings. The chemical structures of the synthesized dyads were characterized by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. The influence of the S/Se atoms and vinyl linkage between the organic unit and C60 on the absorptive and emissive properties of the dyads was investigated and correlated with their photosensitizing activity. For the latter, we used a standard chemical singlet oxygen trap. A selected dyad C60ThSe2 was also applied as a source of singlet oxygen in a model photocatalyzed synthesis of the fine chemical juglone from 1,5-dihydroxynapthalene.

Carbon Quantum Dots from Amino Acids Revisited: Survey of Renewable Precursors toward High Quantum-Yield Blue and Green Fluorescence.

Carbon quantum dots (CQDs) were synthesized via a green, one-step hydrothermal method. As CQD precursors, nine amino acids of different structural descriptors (negatively/positively charged in water, polar, hydrophobic, sulfur-containing, and other/complex ones) were surveyed: Asp, Cys, Gly, His, Leu, Lys, Phe, Pro, and Ser. The reactions were performed in an autoclave in the presence of citric acid at 180 °C for 24 h and yielded core-shell CQDs. CQDs were comprehensively characterized by transmission electron microscopy, dynamic light scattering, Raman, UV/Vis, infrared, X-ray photoelectron spectroscopy, and fluorescence spectroscopy. At the excitation wavelength of λex = 350 nm, Cys-, Phe-, Leu-, and Lys-based CQDs displayed the highest quantum yield blue fluorescence-90 ± 5, 90 ± 4, 87 ± 5, and 67 ± 3%, respectively-superior to the conventional fluorescent dyes. Strikingly, for Lys- and Phe-CQDs, dissimilar trends in the excitation-emission wavelength relationships were identified, that is, constantly strong red shifts versus excitation wavelength-independent emission. Cys- and Lys-CQDs were water-dispersible toward the narrow unimodal distribution of hydrodynamic diameters-0.6 and 2.5 nm, respectively. Additionally, Lys- and Cys-CQDs, with high absolute zeta potential values, formed stable aqueous colloids in a broad range of pH (2, 7, and 12). The results constitute important premises for water-based applications of CQDs, such as bioimaging or photocatalysis.

Mitochondrial Sirtuin-3 (SIRT3) Prevents Doxorubicin-Induced Dilated Cardiomyopathy by Modulating Protein Acetylation and Oxidative Stress.

BACKGROUND: High doses of doxorubicin put cancer patients at risk for developing dilated cardiomyopathy. Previously, we showed that doxorubicin treatment decreases SIRT3 (sirtuin 3), the main mitochondrial deacetylase and increases protein acetylation in rat cardiomyocytes. Here, we hypothesize that SIRT3 expression can attenuate doxorubicin induced dilated cardiomyopathy in vivo by preventing the acetylation of mitochondrial proteins. METHODS: Nontransgenic, M3-SIRT3 (truncated SIRT3; short isoform), and M1-SIRT3 (full-length SIRT3; mitochondrial localized) transgenic mice were treated with doxorubicin for 4 weeks (8 mg/kg body weight per week). Echocardiography was performed to assess cardiac structure and function and validated by immunohistochemistry and immunofluorescence (n=4-10). Mass spectrometry was performed on cardiac mitochondrial peptides in saline (n=6) and doxorubicin (n=5) treated hearts. Validation was performed in doxorubicin treated primary rat and human induced stem cell derived cardiomyocytes transduced with adenoviruses for M3-SIRT3 and M1-SIRT3 and deacetylase deficient mutants (n=4-10). RESULTS: Echocardiography revealed that M3-SIRT3 transgenic mice were partially resistant to doxorubicin induced changes to cardiac structure and function whereas M1-SIRT3 expression prevented cardiac remodeling and dysfunction. In doxorubicin hearts, 37 unique acetylation sites on mitochondrial proteins were altered. Pathway analysis revealed these proteins are involved in energy production, fatty acid metabolism, and oxidative stress resistance. Increased M1-SIRT3 expression in primary rat and human cardiomyocytes attenuated doxorubicin-induced superoxide formation, whereas deacetylase deficient mutants were unable to prevent oxidative stress. CONCLUSIONS: Doxorubicin reduced SIRT3 expression and markedly affected the cardiac mitochondrial acetylome. Increased M1-SIRT3 expression in vivo prevented doxorubicin-induced cardiac dysfunction, suggesting that SIRT3 could be a potential therapeutic target for mitigating doxorubicin-induced dilated cardiomyopathy.

Cardioprotective factors against myocardial infarction selected in vivo from an AAV secretome library.

Therapies for patients with myocardial infarction and heart failure are urgently needed, in light of the breadth of these conditions and lack of curative treatments. To systematically identify previously unidentified cardioactive biologicals in an unbiased manner in vivo, we developed cardiac FunSel, a method for the systematic, functional selection of effective factors using a library of 1198 barcoded adeno-associated virus (AAV) vectors encoding for the mouse secretome. By pooled vector injection into the heart, this library was screened to functionally select for factors that confer cardioprotection against myocardial infarction. After two rounds of iterative selection in mice, cardiac FunSel identified three proteins [chordin-like 1 (Chrdl1), family with sequence similarity 3 member C (Fam3c), and Fam3b] that preserve cardiomyocyte viability, sustain cardiac function, and prevent pathological remodeling. In particular, Chrdl1 exerted its protective activity by binding and inhibiting extracellular bone morphogenetic protein 4 (BMP4), which resulted in protection against cardiomyocyte death and induction of autophagy in cardiomyocytes after myocardial infarction. Chrdl1 also inhibited fibrosis and maladaptive cardiac remodeling by binding transforming growth factor-ß (TGF-ß) and preventing cardiac fibroblast differentiation into myofibroblasts. Production of secreted and circulating Chrdl1, Fam3c, and Fam3b from the liver also protected the heart from myocardial infarction, thus supporting the use of the three proteins as recombinant factors. Together, these findings disclose a powerful method for the in vivo, unbiased selection of tissue-protective factors and describe potential cardiac therapeutics.

Polyamine-Oligonucleotide Conjugates: 2'-OMe-Triazole-Linked 1,4,7,10-Tetraazacyclododecane and Intercalating Dyes and Their Effect on the Thermal Stability of DNA Duplexes.

Oligonucleotides with the sequences 5'-GTG AUPA TGC, 5'-GCA TAUP CAC and 5'-GUPG ATA UPGC, where UP is 2'-O-propargyl uridine, were subjected to post-synthetic Cu(I)-catalyzed azide-alkyne cycloaddition to attach 1,4,7,10-tetraazacyclododecane (cyclen) and two well-known DNA intercalating dyes: thioxanthone and 1,8-naphthalimide. We propose a convenient cyclen protection-deprotection strategy that allows efficient separation of the resulting polyamine-oligonucleotide conjugates from the starting materials by RP-HPLC to obtain high-purity products. In this paper, we present hitherto unknown macrocyclic polyamine-oligonucleotide conjugates and their hybridization properties reflected in the thermal stability of thirty-two DNA duplexes containing combinations of labeled strands, their unmodified complementary strands, and strands with single base pair mismatches. Circular dichroism measurements showed that the B-conformation is retained for all dsDNAs consisting of unmodified and modified oligonucleotides. An additive and destabilizing effect of cyclen moieties attached to dsDNAs was observed. Tm measurements indicate that placing the hydrophobic dye opposite to the cyclen moiety can reduce its destabilizing effect and increase the thermal stability of the duplex. Interestingly, the cyclen-modified U showed significant selectivity for TT mismatch, which resulted in stabilization of the duplex. We conclude the paper with a brief review and discussion in which we compare our results with several examples of oligonucleotides labeled with polyamines at internal strand positions known in the literature.

Age-Dependent Dysregulation of Muscle Vasculature and Blood Flow Recovery after Hindlimb Ischemia in the mdx Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD), caused by a lack of functional dystrophin, is characterized by progressive muscle degeneration. Interestingly, dystrophin is also expressed in endothelial cells (ECs), and insufficient angiogenesis has already been hypothesized to contribute to DMD pathology, however, its status in mdx mice, a model of DMD, is still not fully clear. Our study aimed to reveal angiogenesis-related alterations in skeletal muscles of mdx mice compared to wild-type (WT) counterparts. By investigating 6- and 12-week-old mice, we sought to verify if those changes are age-dependent. We utilized a broad spectrum of methods ranging from gene expression analysis, flow cytometry, and immunofluorescence imaging to determine the level of angiogenic markers and to assess muscle blood vessel abundance. Finally, we implemented the hindlimb ischemia (HLI) model, more biologically relevant in the context of functional studies evaluating angiogenesis/arteriogenesis processes. We demonstrated that both 6- and 12-week-old dystrophic mice exhibited dysregulation of several angiogenic factors, including decreased vascular endothelial growth factor A (VEGF) in different muscle types. Nonetheless, in younger, 6-week-old mdx animals, neither the abundance of CD31+α-SMA+ double-positive blood vessels nor basal blood flow and its restoration after HLI was affected. In 12-week-old mdx mice, although a higher number of CD31+α-SMA+ double-positive blood vessels and an increased percentage of skeletal muscle ECs were found, the abundance of pericytes was diminished, and blood flow was reduced. Moreover, impeded perfusion recovery after HLI associated with a blunted inflammatory and regenerative response was evident in 12-week-old dystrophic mice. Hence, our results reinforce the hypothesis of age-dependent angiogenic dysfunction in dystrophic mice. In conclusion, we suggest that older mdx mice constitute an appropriate model for preclinical studies evaluating the effectiveness of vascular-based therapies aimed at the restoration of functional angiogenesis to mitigate DMD severity.

The Cardiac Lipidome in Models of Cardiovascular Disease.

Cardiovascular disease (CVD) is the leading cause of death worldwide. There are numerous factors involved in the development of CVD. Among these, lipids have an important role in maintaining the myocardial cell structure as well as cardiac function. Fatty acids (FA) are utilized for energy, but also contribute to the pathogenesis of CVD and heart failure. Advances in mass spectrometry methods have enabled the comprehensive analysis of a plethora of lipid species from a single sample comprised of a heterogeneous population of lipid molecules. Determining cardiac lipid alterations in different models of CVD identifies novel biomarkers as well as reveals molecular mechanisms that underlie disease development and progression. This information could inform the development of novel therapeutics in the treatment of CVD. Herein, we provide a review of recent studies of cardiac lipid profiles in myocardial infarction, obesity, and diabetic and dilated cardiomyopathy models of CVD by methods of mass spectrometry analysis.

Characterization of hepatic macrophages and evaluation of inflammatory response in heme oxygenase-1 deficient mice exposed to scAAV9 vectors.

Adeno-associated viral (AAV) vectors are characterised by low immunogenicity, although humoral and cellular responses may be triggered upon infection. Following systemic administration high levels of vector particles accumulate within the liver. Kupffer cells (KCs) are liver resident macrophages and an important part of the liver innate immune system. Decreased functional activity of KCs can contribute to exaggerated inflammatory response upon antigen exposure. Heme oxygenase-1 (HO-1) deficiency is associated with considerably reduced numbers of KCs. In this study we aimed to investigate the inflammatory responses in liver and to characterise two populations of hepatic macrophages in adult wild type (WT) and HO-1 knockout (KO) mice following systemic administration of one or two doses (separated by 3 months) of self-complementary (sc)AAV9 vectors. At steady state, the livers of HO-1 KO mice contained significantly higher numbers of monocyte-derived macrophages (MDMs), but significantly less KCs than their WT littermates. Three days after re-administration of scAAV9 we observed increased mRNA level of monocyte chemoattractant protein-1 (Mcp-1) in the livers of both WT and HO-1 KO mice, but the protein level and the macrophage infiltration were not affected. Three days after the 1st and 3 days after the 2nd vector dose the numbers of AAV genomes in the liver were comparable between both genotypes indicating similar transduction efficiency, but the percentage of transgene-expressing MDMs and KCs was higher in WT than in HO-1 KO mice. In the primary culture, KCs were able to internalize AAV9 particles without induction of TLR9-mediated immune responses, but no transgene expression was observed. In conclusion, in vivo and in vitro cultured KCs have different susceptibility to scAAV9 vectors. Regardless of the presence or absence of HO-1 and initial numbers of KCs in the liver, scAAV9 exhibits a low potential to stimulate inflammatory response at the analysed time points.