Engineering Hydroxylase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of a Key Intermediate to Belzutifan.

Biocatalytic oxidations are an emerging technology for selective C-H bond activation. While promising for a range of selective oxidations, practical use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering and practical application of a non-heme iron and α-ketoglutarate-dependent dioxygenase for the direct stereo- and regio-selective hydroxylation of a non-native fluoroindanone en route to the oncology treatment belzutifan, replacing a five-step chemical synthesis with a direct enantioselective hydroxylation. Mechanistic studies indicated that formation of the desired product was limited by enzyme stability and product overoxidation, with these properties subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. Highlighting the industrial utility of this biocatalyst, the high-yielding, green, and efficient oxidation was demonstrated at kilogram scale for the synthesis of belzutifan.

Head-to-head comparison of in vitro and in vivo efficacy of pHLIP-conjugated anti-seed gamma peptide nucleic acids.

Gamma peptide nucleic acids (γPNAs) have recently garnered attention in diverse therapeutic and diagnostic applications. Serine and diethylene-glycol-containing γPNAs have been tested for numerous RNA-targeting purposes. Here, we comprehensively evaluated the in vitro and in vivo efficacy of pH-low insertion peptide (pHLIP)-conjugated serine and diethylene-based γPNAs. pHLIP targets only the acidic tumor microenvironment and not the normal cells. We synthesized and parallelly tested pHLIP-serine γPNAs and pHLIP-diethylene glycol γPNAs that target the seed region of microRNA-155, a microRNA that is upregulated in various cancers. We performed an all-atom molecular dynamics simulation-based computational study to elucidate the interaction of pHLIP-γPNA constructs with the lipid bilayer. We also determined the biodistribution and efficacy of the pHLIP constructs in the U2932-derived xenograft model. Overall, we established that the pHLIP-serine γPNAs show superior results in vivo compared with the pHLIP-diethylene glycol-based γPNA.

Deficiency of endothelial sirtuin1 in mice stimulates skeletal muscle insulin sensitivity by modifying the secretome.

Downregulation of endothelial Sirtuin1 (Sirt1) in insulin resistant states contributes to vascular dysfunction. Furthermore, Sirt1 deficiency in skeletal myocytes promotes insulin resistance. Here, we show that deletion of endothelial Sirt1, while impairing endothelial function, paradoxically improves skeletal muscle insulin sensitivity. Compared to wild-type mice, male mice lacking endothelial Sirt1 (E-Sirt1-KO) preferentially utilize glucose over fat, and have higher insulin sensitivity, glucose uptake, and Akt signaling in fast-twitch skeletal muscle. Enhanced insulin sensitivity of E-Sirt1-KO mice is transferrable to wild-type mice via the systemic circulation. Endothelial Sirt1 deficiency, by inhibiting autophagy and activating nuclear factor-kappa B signaling, augments expression and secretion of thymosin beta-4 (Tß4) that promotes insulin signaling in skeletal myotubes. Thus, unlike in skeletal myocytes, Sirt1 deficiency in the endothelium promotes glucose homeostasis by stimulating skeletal muscle insulin sensitivity through a blood-borne mechanism, and augmented secretion of Tß4 by Sirt1-deficient endothelial cells boosts insulin signaling in skeletal muscle cells.

SUMOylation of the cardiac sodium channel NaV1.5 modifies inward current and cardiac excitability.

BACKGROUND: Decreased peak sodium current (INa) and increased late sodium current (INa,L), through the cardiac sodium channel NaV1.5 encoded by SCN5A, cause arrhythmias. Many NaV1.5 posttranslational modifications have been reported. A recent report concluded that acute hypoxia increases INa,L by increasing a small ubiquitin-like modifier (SUMOylation) at K442-NaV1.5. OBJECTIVE: The purpose of this study was to determine whether and by what mechanisms SUMOylation alters INa, INa,L, and cardiac electrophysiology. METHODS: SUMOylation of NaV1.5 was detected by immunoprecipitation and immunoblotting. INa was measured by patch clamp with/without SUMO1 overexpression in HEK293 cells expressing wild-type (WT) or K442R-NaV1.5 and in neonatal rat cardiac myocytes (NRCMs). SUMOylation effects were studied in vivo by electrocardiograms and ambulatory telemetry using Scn5a heterozygous knockout (SCN5A+/-) mice and the de-SUMOylating protein SENP2 (AAV9-SENP2), AAV9-SUMO1, or the SUMOylation inhibitor anacardic acid. NaV1.5 trafficking was detected by immunofluorescence. RESULTS: NaV1.5 was SUMOylated in HEK293 cells, NRCMs, and human heart tissue. HyperSUMOylation at NaV1.5-K442 increased INa in NRCMs and in HEK cells overexpressing WT but not K442R-Nav1.5. SUMOylation did not alter other channel properties including INa,L. AAV9-SENP2 or anacardic acid decreased INa, prolonged QRS duration, and produced heart block and arrhythmias in SCN5A+/- mice, whereas AAV9-SUMO1 increased INa and shortened QRS duration. SUMO1 overexpression enhanced membrane localization of NaV1.5. CONCLUSION: SUMOylation of K442-Nav1.5 increases peak INa without changing INa,L, at least in part by altering membrane abundance. Our findings do not support SUMOylation as a mechanism for changes in INa,L. Nav1.5 SUMOylation may modify arrhythmic risk in disease states and represents a potential target for pharmacologic manipulation.

Role of miR-204 in segmental cardiac effects of phenylephrine and pressure overload.

Cardiotoxicity caused by adrenergic receptor agonists overdosing or stress-induced catecholamine release promotes cardiomyopathy, resembling Takotsubo cardiomyopathy (TC). TC is characterized by transient regional systolic dysfunction of the left ventricle. The animal models of TC and modalities for assessing regional wall motion abnormalities in animal models are lacking. We previously reported the protective role of a small noncoding microRNA-204-5p (miR-204) in cardiomyopathies, but its role in TC remains unknown. Here we compared the impact of miR-204 absence on phenylephrine (PE)-induced and transaortic constriction (TAC)-induced changes in cardiac muscle motion in the posterior and anterior apical, mid, and basal segments of the left ventricle using 2-dimensional speckle-tracking echocardiography (2-STE). Wildtype and miR-204-/- mice were subjected to cardiac stress in the form of PE for four weeks or TAC-induced pressure overload for five weeks. PE treatment increased longitudinal and radial motion in the apex of the left ventricle and shortened the peak motion time of all left ventricle segments. The TAC led to decreased longitudinal and radial motion in the left ventricle segments, and there was no difference in the peak motion time. Compared to wildtype mice, PE-induced peak cardiac muscle motion time in the anterior base of the left ventricle was significantly earlier in the miR-204-/- mice. There was no difference in TAC-induced peak cardiac muscle motion time between wildtype and miR-204-/- mice. Our findings demonstrate that PE and TAC induce regional wall motion abnormalities that 2-STE can detect. It also highlights the role of miR-204 in regulating cardiac muscle motion during catecholamine-induced cardiotoxicity.

"We Need More Practice": Evaluating the Role of Virtual Mock OSCES in the Undergraduate Programme During the COVID Pandemic.

Background: Feedback collated at University College London Medical School (UCLMS) during the COVID pandemic identified how many students felt unprepared for their summative Objective Structured Clinical Examinations (OSCEs) despite attending mock face-to-face OSCEs. The aim of this study was to explore the role of virtual mock OSCES for improving student's sense of preparedness and confidence levels for their summative OSCEs. Methods: All Year 5 students (n=354) were eligible to participate in the virtual mock OSCEs and were sent a pre- and post-survey for completion. Hosted on Zoom in June 2021, each circuit comprised six stations, assessing history taking and communication skills only, in Care of the Older Person, Dermatology, Gynaecology, Paediatrics, Psychiatry and Urology. Results: Two hundred and sixty-six Year 5 students (n=354) participated in the virtual mock OSCEs, with 84 (32%) students completing both surveys. While a statistically significant increase in preparedness was demonstrated, there was no difference in overall confidence levels. In contrast, between specialties, a statistically significant increase in confidence levels was seen in all specialties barring Psychiatry. Despite half of the participants highlighting how the format did not sufficiently represent the summative OSCEs, all expressed interest in having virtual mock OSCEs incorporated into the undergraduate programme. Conclusion: The findings of this study suggest that virtual mock OSCEs have a role in preparing medical students for their summative exams. While this was not reflected in their overall confidence levels, this may be due to a lack of clinical exposure and higher anxiety levels among this cohort of students. Although virtual OSCEs cannot replicate the "in-person" experience, considering the logistical advantages, further research is required on how these sessions can be developed, to support the traditional format of face-to-face mock OSCEs within the undergraduate programme.

Dynamin-Independent Mechanisms of Endocytosis and Receptor Trafficking.

Endocytosis is a fundamental mechanism by which cells perform housekeeping functions. It occurs via a variety of mechanisms and involves many regulatory proteins. The GTPase dynamin acts as a "molecular scissor" to form endocytic vesicles and is a critical regulator among the proteins involved in endocytosis. Some GTPases (e.g., Cdc42, arf6, RhoA), membrane proteins (e.g., flotillins, tetraspanins), and secondary messengers (e.g., calcium) mediate dynamin-independent endocytosis. These pathways may be convergent, as multiple pathways exist in a single cell. However, what determines the specific path of endocytosis is complex and challenging to comprehend. This review summarizes the mechanisms of dynamin-independent endocytosis, the involvement of microRNAs, and factors that contribute to the cellular decision about the specific route of endocytosis.

Metabolic rescue ameliorates mitochondrial encephalo-cardiomyopathy in murine and human iPSC models of Leigh syndrome.

BACKGROUND: Mice with deletion of complex I subunit Ndufs4 develop mitochondrial encephalomyopathy resembling Leigh syndrome (LS). The metabolic derangement and underlying mechanisms of cardio-encephalomyopathy in LS remains incompletely understood. METHODS: We performed echocardiography, electrophysiology, confocal microscopy, metabolic and molecular/morphometric analysis of the mice lacking Ndufs4. HEK293 cells, human iPS cells-derived cardiomyocytes and neurons were used to determine the mechanistic role of mitochondrial complex I deficiency. RESULTS: LS mice develop severe cardiac bradyarrhythmia and diastolic dysfunction. Human-induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) with Ndufs4 deletion recapitulate LS cardiomyopathy. Mechanistically, we demonstrate a direct link between complex I deficiency, decreased intracellular (nicotinamide adenine dinucleotide) NAD+ /NADH and bradyarrhythmia, mediated by hyperacetylation of the cardiac sodium channel NaV 1.5, particularly at K1479 site. Neuronal apoptosis in the cerebellar and midbrain regions in LS mice was associated with hyperacetylation of p53 and activation of microglia. Targeted metabolomics revealed increases in several amino acids and citric acid cycle intermediates, likely due to impairment of NAD+ -dependent dehydrogenases, and a substantial decrease in reduced Glutathione (GSH). Metabolic rescue by nicotinamide riboside (NR) supplementation increased intracellular NAD+ / NADH, restored metabolic derangement, reversed protein hyperacetylation through NAD+ -dependent Sirtuin deacetylase, and ameliorated cardiomyopathic phenotypes, concomitant with improvement of NaV 1.5 current and SERCA2a function measured by Ca2+ -transients. NR also attenuated neuronal apoptosis and microglial activation in the LS brain and human iPS-derived neurons with Ndufs4 deletion. CONCLUSIONS: Our study reveals direct mechanistic explanations of the observed cardiac bradyarrhythmia, diastolic dysfunction and neuronal apoptosis in mouse and human induced pluripotent stem cells (iPSC) models of LS.

γ Peptide Nucleic Acid-Based miR-122 Inhibition Rescues Vascular Endothelial Dysfunction in Mice Fed a High-Fat Diet.

The blood levels of microRNA-122 (miR-122) is associated with the severity of cardiovascular disorders, and targeting it with efficient and safer miR inhibitors could be a promising approach. Here, we report the generation of a γ-peptide nucleic acid (γPNA)-based miR-122 inhibitor (γP-122-I) that rescues vascular endothelial dysfunction in mice fed a high-fat diet. We synthesized diethylene glycol-containing γP-122-I and found that its systemic administration counteracted high-fat diet (HFD)-feeding-associated increase in blood and aortic miR-122 levels, impaired endothelial function, and reduced glycemic control. A comprehensive safety analysis established that γP-122-I affects neither the complete blood count nor biochemical tests of liver and kidney functions during acute exposure. In addition, long-term exposure to γP-122-I did not change the overall adiposity, or histology of the kidney, liver, and heart. Thus, γP-122-I rescues endothelial dysfunction without any evidence of toxicity in vivo and demonstrates the suitability of γPNA technology in generating efficient and safer miR inhibitors.

The Enticing Path of miR Therapeutics: Difficult but Not without Prospects.

MicroRNAs (miRs) are short non-coding RNAs that regulate the translation and stability of mRNAs to fine-tune gene expression [...].

The microRNA-204-5p inhibits APJ signalling and confers resistance to cardiac hypertrophy and dysfunction.

BACKGROUND: MicroRNAs regulate cardiac hypertrophy development, which precedes and predicts the risk of heart failure. microRNA-204-5p (miR-204) is well expressed in cardiomyocytes, but its role in developing cardiac hypertrophy and cardiac dysfunction (CH/CD) remains poorly understood. METHODS: We performed RNA-sequencing, echocardiographic, and molecular/morphometric analysis of the heart of mice lacking or overexpressing miR-204 five weeks after trans-aortic constriction (TAC). The neonatal rat cardiomyocytes, H9C2, and HEK293 cells were used to determine the mechanistic role of miR-204. RESULTS: The stretch induces miR-204 expression, and miR-204 inhibits the stretch-induced hypertrophic response of H9C2 cells. The mice lacking miR-204 displayed a higher susceptibility to CH/CD during pressure overload, which was reversed by the adeno-associated virus serotype-9-mediated cardioselective miR-204 overexpression. Bioinformatic analysis of the cardiac transcriptomics of miR-204 knockout mice following pressure overload suggested deregulation of apelin-receptor (APJ) signalling. We found that the stretch-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation and hypertrophy-related genes expression depend on the APJ, and both of these effects are subject to miR-204 levels. The dynamin inhibitor dynasore inhibited both stretch-induced APJ endocytosis and ERK1/2 activation. In contrast, the miR-204-induced APJ endocytosis was neither inhibited by dynamin inhibitors (dynasore and dyngo) nor associated with ERK1/2 activation. We find that the miR-204 increases the expression of ras-associated binding proteins (e.g., Rab5a, Rab7) that regulate cellular endocytosis. CONCLUSIONS: Our results show that miR-204 regulates trafficking of APJ and confers resistance to pressure overload-induced CH/CD, and boosting miR-204 can inhibit the development of CH/CD.

The Challenges and Opportunities in the Development of MicroRNA Therapeutics: A Multidisciplinary Viewpoint.

microRNAs (miRs) are emerging as attractive therapeutic targets because of their small size, specific targetability, and critical role in disease pathogenesis. However, <20 miR targeting molecules have entered clinical trials, and none progressed to phase III. The difficulties in miR target identification, the moderate efficacy of miR inhibitors, cell type-specific delivery, and adverse outcomes have impeded the development of miR therapeutics. These hurdles are rooted in the functional complexity of miR's role in disease and sequence complementarity-dependent/-independent effects in nontarget tissues. The advances in understanding miR's role in disease, the development of efficient miR inhibitors, and innovative delivery approaches have helped resolve some of these hurdles. In this review, we provide a multidisciplinary viewpoint on the challenges and opportunities in the development of miR therapeutics.

Accelerated screening of colloidal nanocrystals using artificial neural network-assisted autonomous flow reactor technology.

Colloidal semiconductor nanocrystals with tunable optical and electronic properties are opening up exciting opportunities for high-performance optoelectronics, photovoltaics, and bioimaging applications. Identifying the optimal synthesis conditions and screening of synthesis recipes in search of efficient synthesis pathways to obtain nanocrystals with desired optoelectronic properties, however, remains one of the major bottlenecks for accelerated discovery of colloidal nanocrystals. Conventional strategies, often guided by limited understanding of the underlying mechanisms remain expensive in both time and resources, thus significantly impeding the overall discovery process. In response, an autonomous experimentation platform is presented as a viable approach for accelerated synthesis screening and optimization of colloidal nanocrystals. Using a machine-learning-based predictive synthesis approach, integrated with automated flow reactor and inline spectroscopy, indium phosphide nanocrystals are autonomously synthesized. Their polydispersity for different target absorption wavelengths across the visible spectrum is simultaneously optimized during the autonomous experimentation, while utilizing minimal self-driven experiments (less than 50 experiments within 2 days). Starting with no-prior-knowledge of the synthesis, an ensemble neural network is trained through autonomous experiments to accurately predict the reaction outcome across the entire synthesis parameter space. The predicted parameter space map also provides new nucleation-growth kinetic insights to achieve high monodispersity in size of colloidal nanocrystals.

Dual-Modality Poly-l-histidine Nanoparticles to Deliver Peptide Nucleic Acids and Paclitaxel for In Vivo Cancer Therapy.

Cationic polymeric nanoformulations have been explored to increase the transfection efficiency of small molecules and nucleic acid-based drugs. However, an excessive positive charge density often leads to severe cell and tissue-based toxicity that restricts the clinical translation of cationic polymeric nanoformulations. Herein, we investigate a series of cationic poly(lactic-co-glycolic acid) (PLGA)-histidine-based nanoformulations for enhanced cytoplasmic delivery with minimal toxicity. PLGA/poly-l-histidine nanoparticles show promising physico-biochemical features and transfection efficiency in a series of in vitro and cell culture-based studies. Further, the use of acetone/dichloromethane as a solvent mixture during the formulation process significantly improves the morphology and size distribution of PLGA/poly-l-histidine nanoparticles. PLGA/poly-l-histidine nanoformulations undergo clathrin-mediated endocytosis. A contrast-matched small-angle neutron scattering experiment confirmed poly-l-histidine's distribution on the PLGA nanoformulations. PLGA/poly-l-histidine formulations containing paclitaxel as a small molecule-based drug and peptide nucleic acids targeting microRNA-155 as nucleic acid analog are efficacious in in vitro and in vivo studies. PLGA/poly-l-histidine NPs significantly decrease tumor growth in PNA-155 (∼6 fold) and paclitaxel (∼6.5 fold) treatment groups in a lymphoma cell line derived xenograft mice model without inducing any toxicity. Hence, PLGA/poly-l-histidine nanoformulations exhibit substantial transfection efficiency and are safe to deliver reagents ranging from small molecules to synthetic nucleic acid analogs and can serve as a novel platform for drug delivery.

Therapeutic Potential of Chemically Modified, Synthetic, Triplex Peptide Nucleic Acid-Based Oncomir Inhibitors for Cancer Therapy.

miRNA-155 (miR-155) is overexpressed in various types of lymphomas and leukemias, suggesting that targeting miR-155 could be a potential platform for the development of precision medicine. Here, we tested the anticancer activity of novel, chemically modified, triplex peptide nucleic acid (PNA)-based antimiRs compared with the current state-of-the-art conventional full-length antimiRs. Next-generation modified PNAs that bound miR-155 by Watson-Crick and Hoogsteen domains possessed superior therapeutic efficacy in vivo and ex vivo compared with conventional full-length anti-miR-155. The efficacy of anti-miR-155 targeting in multiple lymphoma cell lines was comprehensively corroborated by gene expression, Western blot analysis, and cell viability-based functional studies. Finally, preclinical testing in vivo in xenograft mouse models containing lymphoma cell lines demonstrated that treatment with the miR-155-targeting next-generation antimiR resulted in a significant decrease in miR-155 expression, followed by reduced tumor growth. These findings support the effective therapeutic application of chemically modified triplex PNAs to target miR-155 to treat lymphoma. Overall, the present proof-of-concept study further implicates the potential for next-generation triplex gamma PNAs to target other miRNAs for treating cancer. SIGNIFICANCE: This study demonstrates the utility of novel oncomiR inhibitors as cancer therapeutics, providing a new approach for targeting miRNAs and other noncoding RNAs.

Genetic deletion of miR-204 improves glycemic control despite obesity in db/db mice.

MicroRNAs (miRs) are small non-coding RNAs that regulate the target gene expression. A change in miR profile in the pancreatic islets during diabetes is known, and multiple studies have demonstrated that miRs influence the pancreatic ß-cell function. The miR-204 is highly expressed in the ß-cells and reported to regulate insulin synthesis. Here we investigated whether the absence of miR-204 rescues the impaired glycemic control and obesity in the genetically diabetic (db/db) mice. We found that the db/db mice overexpressed miR-204 in the islets. The db/db mice lacking miR-204 (db/db-204-/-) initially develops hyperglycemia and obesity like the control (db/db) mice but later displayed a gradual improvement in glycemic control despite remaining obese. The db/db-204-/- mice had a lower fasting blood glucose and higher serum insulin level compared to the db/db mice. A homeostatic model assessment (HOMA) suggests the improvement of ß-cell function contributes to the improvement in glycemic control in db/db-204-/- mice. Next, we examined the cellular proliferation and endoplasmic reticulum (ER) stress and found an increased frequency of proliferating cells (PCNA + ve) and a decreased CHOP expression in the islets of db/db-204-/- mice. Next, we determined the effect of systemic miR-204 inhibition in improving glycemic control in the high-fat diet (HFD)-fed insulin-resistant mice. MiR-204 inhibition for 6 weeks improved the HFD-triggered impairment in glucose disposal. In conclusion, the absence of miR-204 improves ß-cell proliferation, decreases islet ER stress, and improves glycemic control with limited change in body weight in obese mice.

Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development.

Antisense oligonucleotides (ASOs) bind sequence specifically to the target RNA and modulate protein expression through several different mechanisms. The ASO field is an emerging area of drug development that targets the disease source at the RNA level and offers a promising alternative to therapies targeting downstream processes. To translate ASO-based therapies into a clinical success, it is crucial to overcome the challenges associated with off-target side effects and insufficient biological activity. In this regard, several chemical modifications and diverse delivery strategies have been explored. In this review, we systematically discuss the chemical modifications, mechanism of action, and optimized delivery strategies of several different classes of ASOs. Further, we highlight the recent advances made in development of ASO-based drugs with a focus on drugs that are approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for clinical applications. We also discuss various promising ASO-based drug candidates in the clinical trials, and the outstanding opportunity of emerging microRNA as a viable therapeutic target for future ASO-based therapies.

Microbiota-governed microRNA-204 impairs endothelial function and blood pressure decline during inactivity in db/db mice.

An impaired decline in blood pressure at rest is typical in people with diabetes, reflects endothelial dysfunction, and increases the risk of end-organ damage. Here we report that microRNA-204 (miR-204) promotes endothelial dysfunction and impairment in blood pressure decline during inactivity. We show that db/db mice overexpress miR-204 in the aorta, and its absence rescues endothelial dysfunction and impaired blood pressure decline during inactivity despite obesity. The vascular miR-204 is sensitive to microbiota, and microbial suppression reversibly decreases aortic miR-204 and improves endothelial function, while the endothelial function of mice lacking miR-204 remained indifferent to the microbial alterations. We also show that the circulating miR-122 regulates vascular miR-204 as miR-122 inhibition decreases miR-204 in endothelial cells and aorta. This study establishes that miR-204 impairs endothelial function, promotes impairment in blood pressure decline during rest, and opens avenues for miR-204 inhibition strategies against vascular dysfunction.

SUMO2 regulates vascular endothelial function and oxidative stress in mice.

SUMOylation is a posttranslational modification of lysine residues. Modification of proteins by small ubiquitin-like modifiers (SUMO)1, -2, and -3 can achieve varied, and often unique, physiological and pathological effects. We looked for SUMO2-specific effects on vascular endothelial function. SUMO2 expression was upregulated in the aortic endothelium of hypercholesterolemic low-density lipoprotein receptor-deficient mice and was responsible for impairment of endothelium-dependent vasorelaxation in these mice. Moreover, overexpression of SUMO2 in aortas ex vivo, in cultured endothelial cells, and transgenically in the endothelium of mice increased vascular oxidative stress and impaired endothelium-dependent vasorelaxation. Conversely, inhibition of SUMO2 impaired physiological endothelium-dependent vasorelaxation in normocholesterolemic mice. These findings indicate that while endogenous SUMO2 is important in maintenance of normal endothelium-dependent vascular function, its upregulation impairs vascular homeostasis and contributes to hypercholesterolemia-induced endothelial dysfunction.NEW & NOTEWORTHY Sumoylation is known to impair vascular function; however, the role of specific SUMOs in the regulation of vascular function is not known. Using multiple complementary approaches, we show that hyper-SUMO2ylation impairs vascular endothelial function and increases vascular oxidative stress, whereas endogenous SUMO2 is essential for maintenance of normal physiological function of the vascular endothelium.