Redox-active endosomes mediate α5ß1 integrin signaling and promote chondrocyte matrix metalloproteinase production in osteoarthritis.

Mechanical cues sensed by integrins induce cells to produce proteases to remodel the extracellular matrix. Excessive protease production occurs in many degenerative diseases, including osteoarthritis, in which articular cartilage degradation is associated with the genesis of matrix protein fragments that can activate integrins. We investigated the mechanisms by which integrin signals may promote protease production in response to matrix changes in osteoarthritis. Using a fragment of the matrix protein fibronectin (FN) to activate the α5ß1 integrin in primary human chondrocytes, we found that endocytosis of the integrin and FN fragment complex drove the production of the matrix metalloproteinase MMP-13. Activation of α5ß1 by the FN fragment, but not by intact FN, was accompanied by reactive oxygen species (ROS) production initially at the cell surface, then in early endosomes. These ROS-producing endosomes (called redoxosomes) contained the integrin-FN fragment complex, the ROS-producing enzyme NADPH oxidase 2 (NOX2), and SRC, a redox-regulated kinase that promotes MMP-13 production. In contrast, intact FN was endocytosed and trafficked to recycling endosomes without inducing ROS production. Articular cartilage from patients with osteoarthritis showed increased amounts of SRC and the NOX2 complex component p67phox. Furthermore, we observed enhanced localization of SRC and p67phox at early endosomes, suggesting that redoxosomes could transmit and sustain integrin signaling in response to matrix damage. This signaling mechanism not only amplifies the production of matrix-degrading proteases but also establishes a self-perpetuating cycle that contributes to the ongoing degradation of cartilage matrix in osteoarthritis.

Vitamin B12 does not increase cell viability after hydrogen peroxide induced damage in mouse kidney proximal tubular cells and brain endothelial cells.

Vitamin B12 (B12) is an essential co-factor for two enzymes in mammalian metabolism and can also act as a mimetic of superoxide dismutase (SOD) converting superoxide (O2 •‒) to hydrogen peroxide (H2O2). High oral dose B12 decreases renal O2 •‒ and post-ischemia/reperfusion injury in mice and protects against damage induced by hypoxia/reperfusion in mouse kidney proximal tubular cells (BU.MPT). O2 •‒ is unstable and rapidly converted to H2O2. H2O2 mediates oxidative stress associated with O2 •‒. Whether B12 protects against damage induced by H2O2 is unknown. Both BU.MPT cells and mouse brain endothelial cells (bEdn.3) were applied to test the effects of B12 on H2O2-induced cytotoxicity. Both types of cells were treated with different doses of H2O2 with or without different doses of B12. Cell viability was analyzed 24 h later. H2O2 caused cell death only at a very high dose, and high pharmacological dose of B12 did not prevent this detrimental effect in either cell type. In bEnd.3 cells, transcriptional levels of heme oxygenase-1 (HO-1) increased, while nuclear factor erythroid 2-related factor 2 (Nrf2) decreased by H2O2. The levels of transcripts were not affected by the B12 treatment. We conclude that the cytotoxic effects of exogenous H2O2 in BU.MPT and bEdn.3 cells are not prevented by B12.

Delivery of Cinnamic Aldehyde Antioxidant Response Activating nanoParticles (ARAPas) for Vascular Applications.

Selective delivery of nuclear factor erythroid 2-related factor 2 (Nrf2) activators to the injured vasculature at the time of vascular surgical intervention has the potential to attenuate oxidative stress and decrease vascular smooth muscle cell (VSMC) hyperproliferation and migration towards the inner vessel wall. To this end, we developed a nanoformulation of cinnamic aldehyde (CA), termed Antioxidant Response Activating nanoParticles (ARAPas), that can be readily loaded into macrophages ex vivo. The CA-ARAPas-macrophage system was used to study the effects of CA on VSMC in culture. CA was encapsulated into a pluronic micelle that was readily loaded into both murine and human macrophages. CA-ARAPas inhibits VSMC proliferation and migration, and activates Nrf2. Macrophage-mediated transfer of CA-ARAPas to VSMC is evident after 12 h, and Nrf2 activation is apparent after 24 h. This is the first report, to the best of our knowledge, of CA encapsulation in pluronic micelles for macrophage-mediated delivery studies. The results of this study highlight the feasibility of CA encapsulation and subsequent macrophage uptake for delivery of cargo into other pertinent cells, such as VSMC.

Allyship in Surgical Residents: Evidence for LGBTQ Competency Training in Surgical Education.

BACKGROUND: Studies have shown poorer health outcomes for people who identify as sexual and/or gender minority (LGBTQ+) compared to heterosexual peers. Our goal was to establish baseline levels of LGBTQ Ally Identity Measure (AIM) scores: (1) Knowledge and Skills, (2) Openness and Support, and (3) Awareness of Oppression of the LGBTQ+ in surgical trainees, and implement a pilot training in LGBTQ + cultural competency. MATERIALS AND METHODS: General surgery residents from a single academic medical center participated in a 2-h educational training developed from the existing Health Care Safe Zone training at our institution. Utilizing the previously validated LGBTQ Ally Identity Measure (AIM), residents responded to 19 items on Likert-type scales from 1 to 5 pretraining and 6 wk posttraining. The residents' perceptions of the utility of the training were also assessed. Data were analyzed by MANOVA, repeated measures MANOVA, and subsequent univariate analysis. RESULTS: 27 residents responded to the pretraining survey (52%), 22 residents participated in the training, and 10 responded at 6 wk posttraining (19%). The average baseline scores were Knowledge and Skills 19.38 ± 4.64, Openness and Support 25.96 ± 4.31, and Awareness of Oppression 17.15 ± 2.20. Participants who identified as women scored 4.46 (95% CI 0.77-8.15) points higher in Openness and Support compared to males. Of those respondents who completed pretraining and posttraining surveys (n = 10), training had a significant effect on AIM scores with an improvement in Knowledge and Skills (P = 0.024) and Openness and Support (P = 0.042). Residents found the training relevant to surgery patient care (71%), increased their competency in LGBTQ + patient care (86%), and all participants indicated they were better LGBTQ allies following the training. CONCLUSIONS: Assessing LGBTQ + allyship in surgical residents, we found that training improved AIM scores over time with significant improvement in the Knowledge and Skills, and Openness and Support scales, suggesting a viable and valuable curriculum focused on sexual and gender identity-related competencies within the graduate medical education for surgical trainees.

Pharmacokinetics and biodistribution of a collagen-targeted peptide amphiphile for cardiovascular applications.

Atherosclerosis remains a leading cause of death and disability around the world and a major driver of health care spending. Nanomaterials have gained widespread attention due to their promising potential for clinical translation and use. We have developed a collagen-targeted peptide amphiphile (PA)-based nanofiber for the prevention of neointimal hyperplasia after arterial injury. Our goal was to characterize the pharmacokinetics and biodistribution of the collagen-targeted PA to further its advancement into clinical trials. Collagen-targeted PA was injected into the internal jugular vein of Sprague Dawley rats. PA concentrations in plasma collected at various times after injection (0 to 72 hours) were measured by liquid chromatography-tandem mass spectrometry. Pharmacokinetics of the collagen-targeted PA were characterized by a three-compartment model, with an extremely rapid apparent elimination clearance resulting in a plasma concentration decrease of more than two orders of magnitude within the first hour after injection. This rapid initial decline in plasma concentration was not due to degradation by plasma components, as collagen-targeted PA was stable in plasma ex vivo for up to 3 hours. Indeed, cellular blood components appear to be partly responsible, as only 15% of collagen-targeted PA were recovered following incubation with whole blood. Nanofibers in whole blood also adhered to red blood cells (RBCs) and were engulfed by mononuclear cells. This work highlights the unique pharmacokinetics of our collagen-targeted PA, which differ from pharmacokinetics of small molecules. Because of their targeted nature, these nanomaterials should not require sustained elevated plasma concentrations to achieve a therapeutic effect the way small molecules typically do.

Cannabis sativa extracts protect LDL from Cu2+-mediated oxidation.

BACKGROUND: Multiple therapeutic properties have been attributed to Cannabis sativa. However, further research is required to unveil the medicinal potential of Cannabis and the relationship between biological activity and chemical profile. OBJECTIVES: The primary objective of this study was to characterize the chemical profile and antioxidant properties of three varieties of Cannabis sativa available in Uruguay during progressive stages of maturation. METHODS: Fresh samples of female inflorescences from three stable Cannabis sativa phenotypes, collected at different time points during the end of the flowering period were analyzed. Chemical characterization of chloroform extracts was performed by 1H-NMR. The antioxidant properties of the cannabis sativa extracts, and pure cannabinoids, were measured in a Cu2+-induced LDL oxidation assay. RESULTS: The main cannabinoids in the youngest inflorescences were tetrahydrocannabinolic acid (THC-A, 242 ± 62 mg/g) and tetrahydrocannabinol (THC, 7.3 ± 6.5 mg/g). Cannabinoid levels increased more than twice in two of the mature samples. A third sample showed a lower and constant concentration of THC-A and THC (177 ± 25 and 1 ± 1, respectively). The THC-A/THC rich cannabis extracts increased the latency phase of LDL oxidation by a factor of 1.2-3.5 per µg, and slowed down the propagation phase of lipoperoxidation (IC50 1.7-4.6 µg/mL). Hemp, a cannabidiol (CBD, 198 mg/g) and cannabidiolic acid (CBD-A, 92 mg/g) rich variety, also prevented the formation of conjugated dienes during LDL oxidation. In fact, 1 µg of extract was able to stretch the latency phase 3.7 times and also to significantly reduce the steepness of the propagation phase (IC50 of 8 µg/mL). Synthetic THC lengthened the duration of the lag phase by a factor of 21 per µg, while for the propagation phase showed an IC50 ≤ 1 µg/mL. Conversely, THC-A was unable to improve any parameter. Meanwhile, the presence of 1 µg of pure CBD and CBD-A increased the initial latency phase 4.8 and 9.4 times, respectively, but did not have an effect on the propagation phase. CONCLUSION: Cannabis whole extracts acted on both phases of lipid oxidation in copper challenged LDL. Those effects were just partially related with the content of cannabinoids and partially recapitulated by isolated pure cannabinoids. Our results support the potentially beneficial effects of cannabis sativa whole extracts on the initial phase of atherosclerosis.

Diabetic vasculopathy: macro and microvascular injury.

PURPOSE OF REVIEW: Diabetes is a common and prevalent medical condition as it affects many lives around the globe. Specifically, type-2 Diabetes (T2D) is characterized by chronic systemic inflammation alongside hyperglycemia and insulin resistance in the body, which can result in atherosclerotic legion formation in the arteries and thus progression of related conditions called diabetic vasculopathies. T2D patients are especially at risk for vascular injury; adjunct in many of these patients heir cholesterol and triglyceride levels reach dangerously high levels and accumulate in the lumen of their vascular system. RECENT FINDINGS: Microvascular and macrovascular vasculopathies as complications of diabetes can accentuate the onset of organ illnesses, thus it is imperative that research efforts help identify more effective methods for prevention and diagnosis of early vascular injuries. Current research into vasculopathy identification/treatment will aid in the amelioration of diabetes-related symptoms and thus reduce the large number of deaths that this disease accounts annually. SUMMARY: This review aims to showcase the evolution and effects of diabetic vasculopathy from development to clinical disease as macrovascular and microvascular complications with a concerted reference to sex-specific disease progression as well.

Oral high dose vitamin B12 decreases renal superoxide and post-ischemia/reperfusion injury in mice.

Renal ischemia/reperfusion injury (IRI) is a leading cause of acute kidney injury (AKI), a potentially fatal syndrome characterized by a rapid decline in kidney function. Excess production of superoxide contributes to the injury. We hypothesized that oral administration of a high dose of vitamin B12 (B12 - cyanocobalamin), which possesses a superoxide scavenging function, would protect kidneys against IRI and provide a safe means of treatment. Following unilateral renal IR surgery, C57BL/6J wild type (WT) mice were administered B12 via drinking water at a dose of 50 mg/L. After 5 days of the treatment, plasma B12 levels increased by 1.2-1.5x, and kidney B12 levels increased by 7-8x. IRI mice treated with B12 showed near normal renal function and morphology. Further, IRI-induced changes in RNA and protein markers of inflammation, fibrosis, apoptosis, and DNA damage response (DDR) were significantly attenuated by at least 50% compared to those in untreated mice. Moreover, the presence of B12 at 0.3 µM in the culture medium of mouse proximal tubular cells subjected to 3 hr of hypoxia followed by 1 hr of reperfusion in vitro showed similar protective effects, including increased cell viability and decreased reactive oxygen species (ROS) level. We conclude that a high dose of B12 protects against perfusion injury both in vivo and in vitro without observable adverse effects in mice and suggest that B12 merits evaluation as a treatment for I/R-mediated AKI in humans.

Nanotherapies for Treatment of Cardiovascular Disease: A Case for Antioxidant Targeted Delivery.

PURPOSE OF REVIEW: Cardiovascular disease (CVD) involves a broad range of clinical manifestations resulting from a dysfunctional vascular system. Overproduction of reactive oxygen and nitrogen species are causally implicated in the severity of vascular dysfunction and CVD. Antioxidant therapy is an attractive avenue for treatment of CVD associated pathologies. Implementation of targeted nano-antioxidant therapies has the potential to overcome hurdles associated with systemic delivery of antioxidants. This review examines the currently available options for nanotherapeutic targeting CVD, and explores successful studies showcasing targeted nano-antioxidant therapy. RECENT FINDINGS: Active targeting strategies in the context of CVD heavily focus on immunotargeting to inflammatory markers like cell adhesion molecules, or to exposed extracellular matrix components. Targeted antioxidant nanotherapies have found success in pre-clinical studies. SUMMARY: This review underscores the potential of targeted nanocarriers as means of finding success translating antioxidant therapies to the clinic, all with a focus on CVD.

Engulfment and cell motility protein 1 potentiates diabetic cardiomyopathy via Rac-dependent and Rac-independent ROS production.

Engulfment and cell motility protein 1 (ELMO1) is part of a guanine nucleotide exchange factor for Ras-related C3 botulinum toxin substrate (Rac), and ELMO1 polymorphisms were identified to be associated with diabetic nephropathy in genome-wide association studies. We generated a set of Akita Ins2C96Y diabetic mice having 5 graded cardiac mRNA levels of ELMO1 from 30% to 200% of normal and found that severe dilated cardiomyopathy develops in ELMO1-hypermorphic mice independent of renal function at age 16 weeks, whereas ELMO1-hypomorphic mice were completely protected. As ELMO1 expression increased, reactive oxygen species indicators, dissociation of the intercalated disc, mitochondrial fragmentation/dysfunction, cleaved caspase-3 levels, and actin polymerization increased in hearts from Akita mice. Cardiomyocyte-specific overexpression in otherwise ELMO1-hypomorphic Akita mice was sufficient to promote cardiomyopathy. Cardiac Rac1 activity was positively correlated with the ELMO1 levels, and oral administration of a pan-Rac inhibitor, EHT1864, partially mitigated cardiomyopathy of the ELMO1 hypermorphs. Disrupting Nox4, a Rac-independent NADPH oxidase, also partially mitigated it. In contrast, a pan-NADPH oxidase inhibitor, VAS3947, markedly prevented cardiomyopathy. Our data demonstrate that in diabetes mellitus ELMO1 is the "rate-limiting" factor of reactive oxygen species production via both Rac-dependent and Rac-independent NADPH oxidases, which in turn trigger cellular signaling cascades toward cardiomyopathy.

Atheroma Niche-Responsive Nanocarriers for Immunotherapeutic Delivery.

Nanomedicine is a promising, noninvasive approach to reduce atherosclerotic plaque burden. However, drug delivery is limited without the ability of nanocarriers to sense and respond to the diseased microenvironment. In this study, nanomaterials are developed from peptide amphiphiles (PAs) that respond to the increased levels of matrix metalloproteinases 2 and 9 (MMP2/9) or reactive oxygen species (ROS) found within the atherosclerotic niche. A pro-resolving therapeutic, Ac2-26, derived from annexin-A1 protein, is tethered to PAs using peptide linkages that cleave in response to MMP2/9 or ROS. By adjusting the molar ratios and processing conditions, the Ac2-26 PA can be co-assembled with a PA containing an apolipoprotein A1-mimetic peptide to create a targeted, therapeutic nanofiber (ApoA1-Ac226 PA). The ApoA1-Ac2-26 PAs demonstrate release of Ac2-26 within 24 h after treatment with MMP2 or ROS. The niche-responsive ApoA1-Ac2-26 PAs are cytocompatible and reduce macrophage activation from interferon gamma and lipopolysaccharide treatment, evidenced by decreased nitric oxide production. Interestingly, the linkage chemistry of ApoA1-Ac2-26 PAs significantly affects macrophage uptake and retention. Taken together, these findings demonstrate the potential of PAs to serve as an atheroma niche-responsive nanocarrier system to modulate the inflammatory microenvironment, with implications for atherosclerosis treatment.

Inhibiting intimal hyperplasia in prosthetic vascular grafts via immobilized all-trans retinoic acid.

Peripheral arterial disease is a leading cause of morbidity and mortality. The most commonly utilized prosthetic material for peripheral bypass grafting is expanded polytetrafluoroethylene (ePTFE) yet it continues to exhibit poor performance from restenosis due to neointimal hyperplasia, especially in femoral distal bypass procedures. Recently, we demonstrated that periadventitial delivery of all-trans retinoic acid (atRA) immobilized throughout porous poly(1,8 octamethylene citrate) (POC) membranes inhibited neointimal formation in a rat arterial injury model. Thus, the objective of this study was to investigate whether atRA immobilized throughout the lumen of ePTFE vascular grafts would inhibit intimal formation following arterial bypass grafting. Utilizing standard ePTFE, two types of atRA-containing ePTFE vascular grafts were fabricated and evaluated: grafts whereby all-trans retinoic acid was directly immobilized on ePTFE (atRA-ePTFE) and grafts where all-trans retinoic acid was immobilized onto ePTFE grafts coated with POC (atRA-POC-ePTFE). All grafts were characterized by SEM, HPLC, and FTIR and physical characteristics were evaluated in vitro. Modification of these grafts, did not significantly alter their physical characteristics or biocompatibility, and resulted in inhibition of intimal formation in a rat aortic bypass model, with atRA-POC-ePTFE inhibiting intimal formation at both the proximal and distal graft sections. In addition, treatment with atRA-POC-ePTFE resulted in increased graft endothelialization and decreased inflammation when compared to the other treatment groups. This work further confirms the biocompatibility and efficacy of locally delivered atRA to inhibit intimal formation in a bypass setting. Thus, atRA-POC-ePTFE grafts have the potential to improve patency rates in small diameter bypass grafts and warrant further investigation.