Isabel N. Schellinger

Diabetic Cardiovascular Disease Induced by Oxidative Stress

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality among patients with diabetes mellitus (DM). DM can lead to multiple cardiovascular complications, including coronary artery disease (CAD), cardiac hypertrophy, and heart failure (HF). HF represents one of the most common causes of death in patients with DM and results from DM-induced CAD and diabetic cardiomyopathy. Oxidative stress is closely associated with the pathogenesis of DM and results from overproduction of reactive oxygen species (ROS). ROS overproduction is associated with hyperglycemia and metabolic disorders, such as impaired antioxidant function in conjunction with impaired antioxidant activity. Long-term exposure to oxidative stress in DM induces chronic inflammation and fibrosis in a range of tissues, leading to formation and progression of disease states in these tissues. Indeed, markers for oxidative stress are overexpressed in patients with DM, suggesting that increased ROS may be primarily responsible for the development of diabetic complications. Therefore, an understanding of the pathophysiological mechanisms mediated by oxidative stress is crucial to the prevention and treatment of diabetes-induced CVD. The current review focuses on the relationship between diabetes-induced CVD and oxidative stress, while highlighting the latest insights into this relationship from findings on diabetic heart and vascular disease.

Segmental Aortic Stiffening Contributes to Experimental Abdominal Aortic Aneurysm Development

Background— Stiffening of the aortic wall is a phenomenon consistently observed in age and in abdominal aortic aneurysm (AAA). However, its role in AAA pathophysiology is largely undefined. Methods and Results— Using an established murine elastase-induced AAA model, we demonstrate that segmental aortic stiffening precedes aneurysm growth. Finite-element analysis reveals that early stiffening of the aneurysm-prone aortic segment leads to axial (longitudinal) wall stress generated by cyclic (systolic) tethering of adjacent, more compliant wall segments. Interventional stiffening of AAA-adjacent aortic segments (via external application of surgical adhesive) significantly reduces aneurysm growth. These changes correlate with the reduced segmental stiffness of the AAA-prone aorta (attributable to equalized stiffness in adjacent segments), reduced axial wall stress, decreased production of reactive oxygen species, attenuated elastin breakdown, and decreased expression of inflammatory cytokines and macrophage infiltration, and attenuated apoptosis within the aortic wall, as well. Cyclic pressurization of segmentally stiffened aortic segments ex vivo increases the expression of genes related to inflammation and extracellular matrix remodeling. Finally, human ultrasound studies reveal that aging, a significant AAA risk factor, is accompanied by segmental infrarenal aortic stiffening. Conclusions— The present study introduces the novel concept of segmental aortic stiffening as an early pathomechanism generating aortic wall stress and triggering aneurysmal growth, thereby delineating potential underlying molecular mechanisms and therapeutic targets. In addition, monitoring segmental aortic stiffening may aid the identification of patients at risk for AAA.

Transcription Factor Runx2 Promotes Aortic Fibrosis and Stiffness in Type 2 Diabetes Mellitus

RATIONALE Accelerated arterial stiffening is a major complication of diabetes mellitus with no specific therapy available to date. OBJECTIVE The present study investigates the role of the osteogenic transcription factor runt-related transcription factor 2 (Runx2) as a potential mediator and therapeutic target of aortic fibrosis and aortic stiffening in diabetes mellitus. METHODS AND RESULTS Using a murine model of type 2 diabetes mellitus (db/db mice), we identify progressive structural aortic stiffening that precedes the onset of arterial hypertension. At the same time, Runx2 is aberrantly upregulated in the medial layer of db/db aortae, as well as in thoracic aortic samples from patients with type 2 diabetes mellitus. Vascular smooth muscle cell-specific overexpression of Runx2 in transgenic mice increases expression of its target genes, Col1a1 and Col1a2, leading to medial fibrosis and aortic stiffening. Interestingly, increased Runx2 expression per se is not sufficient to induce aortic calcification. Using in vivo and in vitro approaches, we further demonstrate that expression of Runx2 in diabetes mellitus is regulated via a redox-sensitive pathway that involves a direct interaction of NF-κB with the Runx2 promoter. CONCLUSIONS In conclusion, this study highlights Runx2 as a previously unrecognized inducer of vascular fibrosis in the setting of diabetes mellitus, promoting arterial stiffness irrespective of calcification.

MicroRNA-210 Enhances Fibrous Cap Stability in Advanced Atherosclerotic Lesions

Rationale: In the search for markers and modulators of vascular disease, microRNAs (miRNAs) have emerged as potent therapeutic targets. Objective: To investigate miRNAs of clinical interest in patients with unstable carotid stenosis at risk of stroke. Methods and Results: Using patient material from the BiKE (Biobank of Karolinska Endarterectomies), we profiled miRNA expression in patients with stable versus unstable carotid plaque. A polymerase chain reaction–based miRNA array of plasma, sampled at the carotid lesion site, identified 8 deregulated miRNAs (miR-15b, miR-29c, miR-30c/d, miR-150, miR-191, miR-210, and miR-500). miR-210 was the most significantly downregulated miRNA in local plasma material. Laser capture microdissection and in situ hybridization revealed a distinct localization of miR-210 in fibrous caps. We confirmed that miR-210 directly targets the tumor suppressor gene APC (adenomatous polyposis coli), thereby affecting Wnt (Wingless-related integration site) signaling and regulating smooth muscle cell survival, as well as differentiation in advanced atherosclerotic lesions. Substantial changes in arterial miR-210 were detectable in 2 rodent models of vascular remodeling and plaque rupture. Modulating miR-210 in vitro and in vivo improved fibrous cap stability with implications for vascular disease. Conclusions: An unstable carotid plaque at risk of stroke is characterized by low expression of miR-210. miR-210 contributes to stabilizing carotid plaques through inhibition of APC, ensuring smooth muscle cell survival. We present local delivery of miR-210 as a therapeutic approach for prevention of atherothrombotic vascular events.

MicroRNA-Mediated Therapy Modulating Blood–Brain Barrier Disruption Improves Vascular Cognitive Impairment

Objective— There are currently no effective treatments for the prevention of dementia associated with vascular cognitive impairment. MicroRNAs regulate gene expression at the post-transcriptional level and play key roles in vascular disorders. TNF&agr; (tumor necrosis factor-&agr;) regulates blood–brain barrier breakdown through modification of cerebral tight junctions. Here, we sought key TNF&agr;-responsive microRNAs that might influence blood–brain barrier breakdown via cerebral tight junction disruption in vascular cognitive impairment. Approach and Results— Using a mouse model of vascular cognitive impairment, chronic cerebral hypoperfusion within the white matter was induced with bilateral common carotid artery stenosis (BCAS) surgery. TNF&agr; gene expression was increased in white matter post-BCAS surgery, and TNF&agr; stimulation decreased claudin-5, ZO-1 (tight-junction protein 1), and occludin gene expression in murine brain endothelial cells. In silico analysis predicted 8 candidate microRNAs as regulators of claudin-5, ZO-1, and occludin gene expression. Of these, only miR-501-3p was upregulated by TNF&agr; in vitro and was upregulated in the white matter after BCAS surgery. Further, miR-501-3p directly bound to the 3′-untranslated region of human ZO-1 and downregulated transendothelial electric resistance. In vivo administration of a locked nucleic acid –modified antisense oligonucleotide versus miR-501-3p suppressed BCAS-induced reduction of ZO-1 gene expression and blood–brain barrier disruption within the white matter and significantly ameliorated working memory deficits after BCAS surgery. Conclusions— We here provide the first evidence that the TNF&agr;–miR-501-3p–ZO-1 axis plays an important role in the pathogenesis of cerebral hypoperfusion–induced working memory deficits and white matter lesions, as a result of blood–brain barrier breakdown via tight junction disruption. Therapeutic manipulation of miR-501-3p holds promise for limiting vascular cognitive impairment progression.

Systemic Upregulation of IL-10 (Interleukin-10) Using a Nonimmunogenic Vector Reduces Growth and Rate of Dissecting Abdominal Aortic Aneurysm

Objective— Recruitment of immunologic competent cells to the vessel wall is a crucial step in formation of abdominal aortic aneurysms (AAA). Innate immunity effectors (eg, macrophages), as well as mediators of adaptive immunity (eg, T cells), orchestrate a local vascular inflammatory response. IL-10 (interleukin-10) is an immune-regulatory cytokine with a crucial role in suppression of inflammatory processes. We hypothesized that an increase in systemic IL-10-levels would mitigate AAA progression. Approach and Results— Using a single intravenous injection protocol, we transfected an IL-10 transcribing nonimmunogenic minicircle vector into the Ang II (angiotensin II)-ApoE−/− infusion mouse model of AAA. IL-10 minicircle transfection significantly reduced average aortic diameter measured via ultrasound at day 28 from 166.1±10.8% (control) to 131.0±5.8% (IL-10 transfected). Rates of dissecting AAA were reduced by IL-10 treatment, with an increase in freedom from dissecting AAA from 21.5% to 62.3%. Using flow cytometry of aortic tissue from minicircle IL-10-treated animals, we found a significantly higher percentage of CD4+/CD25+/Foxp3 (forkhead box P3)+ regulatory T cells, with fewer CD8+/GZMB+ (granzyme B) cytotoxic T cells. Furthermore, isolated aortic macrophages produced less TNF-&agr; (tumor necrosis factor-&agr;), more IL-10, and were more likely to be MRC1 (mannose receptor, C type 1)–positive alternatively activated macrophages. These results concurred with gene expression analysis of lipopolysaccharide-stimulated and Ang II-primed human peripheral blood mononuclear cells. Conclusions— Taken together, we provide an effective gene therapy approach to AAA in mice by enhancing antiinflammatory and dampening proinflammatory pathways through minicircle-induced augmentation of systemic IL-10 expression.