Diabetes is accompanied by secretion of pro-atherosclerotic exosomes from vascular smooth muscle cells.

BACKGROUND: Atherosclerosis is a common co-morbidity of type 2 diabetes mellitus. Monocyte recruitment by an activated endothelium and the pro-inflammatory activity of the resulting macrophages are critical components of atherosclerosis. Exosomal transfer of microRNAs has emerged as a paracrine signaling mechanism regulating atherosclerotic plaque development. MicroRNAs-221 and -222 (miR-221/222) are elevated in vascular smooth muscle cells (VSMCs) of diabetic patients. We hypothesized that the transfer of miR-221/222 via VSMC-derived exosomes from diabetic sources (DVEs) promotes increased vascular inflammation and atherosclerotic plaque development. METHODS: Exosomes were obtained from VSMCs, following exposure to non-targeting or miR-221/-222 siRNA (-KD), isolated from diabetic (DVEs) and non-diabetic (NVEs) sources and their miR-221/-222 content was measured using droplet digital PCR (ddPCR). Expression of adhesion molecules and the adhesion of monocytes was measured following exposure to DVEs and NVEs. Macrophage phenotype following exposure to DVEs was determined by measuring mRNA markers and secreted cytokines. Age-matched apolipoprotein-E-deficient mice null (ApoE-/-) mice were maintained on Western diet for 6 weeks and received injections of saline, NVEs, NVE-KDs, DVEs or DVE-KDs every other day. Atherosclerotic plaque formation was measured using Oil Red Oil staining. RESULTS: Exposure of human umbilical vein and coronary artery endothelial cells to DVEs, but not NVEs, NVE-KDs, or DVE-KDs promoted increased intercellular adhesion molecule-1 expression and monocyte adhesion. DVEs but not NVEs, NVE-KDs, or DVE-KDs also promoted pro-inflammatory polarization of human monocytes in a miR-221/222 dependent manner. Finally, intravenous administration of DVEs, but not NVEs, resulted in a significant increase in atherosclerotic plaque development. CONCLUSION: These data identify a novel paracrine signaling pathway that promotes the cardiovascular complications of diabetes mellitus.

Hydrophobic Patch of Ubiquitin is Important for its Optimal Activation by Ubiquitin Activating Enzyme E1.

Protein ubiquitination plays a role in essentially every process in eukaryotic cells. The attachment of ubiquitin (Ub) or Ub-like (UBL) proteins to target proteins is achieved by parallel but distinct cascades of enzymatic reactions involving three enzymes: E1, E2, and E3. The E1 enzyme functions at the apex of this pathway and plays a critical role in activating the C-terminus of ubiquitin or UBL, which is an essential step that triggers subsequent downstream transfer to their cognate E2s resulting in the fidelity of the Ub/UBL conjugation machinery. Despite the central role of the E1 enzyme in protein modification, a quantitative method to measure Ub/UBL activation by E1 is lacking. Here, we present a mass spectrometry-based assay to accurately measure the activation of Ub/UBL by E1 independent of the E2/E3 enzymes. Our method does not require radiolabeling of any components and therefore can be used in any biochemical laboratory having access to a mass spectrometer. This method allowed us to dissect the concerted process of E1-E2-catalyzed Ub conjugation in order to separately characterize the process of Ub activation and how it is affected by select mutations and other factors. We found that the hydrophobic patch of Ub is important for the optimal activation of Ub by E1. We further show that the blockers of the Ub-proteasome system such as ubistatin and fullerenol inhibit Ub activation by E1. Interestingly, our data indicate that the phosphorylation of Ub at the S65 position augments its activation by the E1 enzyme.

Intraoperative neurophysiologic monitoring prevented iatrogenic spinal cord injury during robotic-assisted transabdominal adrenalectomy: a case report.

Cervical spondylotic myelopathy (CSM) is the most common disease of the cervical spinal cord in patients older than 55 and is characterized by an initial asymptomatic period followed by progressive neurological deficit from degenerative changes of the cervical vertebrae. These changes cause compression and vascular compromise to the cervical spinal cord. Because there are no pathognomonic symptoms, its diagnosis is commonly delayed. Herein we report the first case of the use of IONM during a transabdominal adrenalectomy in a patient with CSM, which prevented an iatrogenic spinal cord injury (SCI). The patient is a 74-year-old male with what was proven later as cervical spinal stenosis who presented for robotic-assisted transabdominal adrenalectomy. When positioned supine on the operating table, he exhibited upper and lower extremity neurological symptoms, prompting awake fiberoptic intubation and the use of IONM secondary to suspicion for CSM. After being positioned into lateral decubitus, IONM showed a loss of transcranial motor evoked potentials (TcMEP) and attenuated somatosensory evoked potentials (SSEP) from the right lower extremities and the procedure was aborted and the patient returned supine. TcMEPs returned to baseline, but SSEPs remained attenuated. The patient exhibited normal movement and sensation in post-anesthesia care. A high index of suspicion for CSM is required for older patients, as early diagnosis allows for spinal surgery treatment before acute worsening during anesthesia or non-spinal surgery. Furthermore, a low threshold for the use of IONM in patients with a high likelihood of CSM who require a non-spinal surgery can successfully prevent iatrogenic SCI.

Modulation of inflammation in wounds of diabetic patients treated with porcine urinary bladder matrix.

Aim: To determine if porcine urinary bladder matrix (UBM) treatment is associated with modulation of wound inflammation in diabetic patients. Patients & methods: mRNA associated with M1 and M2 macrophages were measured in wounds of diabetic and nondiabetic patients pre- and post-treatment with UBM and an M1:M2 score was calculated. Results: Wound tissue from diabetic subjects exhibited elevated M1:M2 scores compared with nondiabetic patients, suggesting a greater pro-inflammatory state prior to treatment. Post-treatment, there was significantly greater reduction in the magnitude of the individual M1:M2 scores in the diabetic patients resulting in similar levels in both groups of patients. Conclusions: UBM may assist in diabetic wound healing by restoring an inflammatory state similar to that of nondiabetic patients.