Aspects of arterial wall healing : re-endothelialization, intimal hyperplasia and vascular remodeling

Cardiovascular disease is the leading cause of mortality in the world. Despite prevention, the need for interventions remains high. Patients with type 2 diabetes mellitus have an increased cardiovascular burden and are at higher risk of complications following invasive vascular interventions. Complications related to an excessive healing response are a major clinical problem, which results in increased morbidity and possibly death. The arterial wall healing response consists of re-endothelialization, intimal hyperplasia (IH) formation and vascular remodeling. Current pharmacological treatment relies on non-selective anti-proliferative drugs, which reduces IH formation but increases the risk of thrombosis due to a delayed re-endothelialization. Hence, there is a need for development of selective treatments. Evaluation of the re-endothelialization process in the rat carotid balloon injury model has previously been limited to histological staining and invasive imaging techniques. We demonstrate that it is possible to estimate the re-endothelialization process in ultrasound biomicroscopy using IH morphology as a surrogate marker. This technique will be a useful tool for non-invasive real-time evaluation of the re-endothelialization process in pharmacological studies. Incretin-modulating drugs is a group of antidiabetic drugs, which targets the glucagon-like peptide-1 (GLP-1) receptor by either direct activation or suppressing breakdown of native GLP-1 with dipeptidylpeptidase-4 (DPP-4) inhibitors. GLP-1 receptor activation has been shown to reduce IH formation by selective inhibition of smooth muscle cell (SMC) proliferation. However, we show that treatment with linagliptin, a DPP-4 inhibitor, does not influence the arterial wall healing in normal or type 2 diabetic conditions. Large-scale transcriptomic analysis is an important tool for confirmation and identification of novel molecular mechanisms in experimental research. In Study III, we generated an encyclopedia of the transcriptomic landscape over time in the rat carotid balloon injury model. We could detect three separate phases of the healing process and contribution of novel molecular mechanisms. This resource includes a biobank of tissue samples, which will be a powerful tool for validation and identification of novel treatment targets. The utilization of transcriptomic data to identify new biological pathways in the arterial wall healing process can be exemplified with proprotein convertase subtilisin/kexin 6 (PCSK6). Previously, we could identify an increased expression of PCSK6 in patients with symptomatic carotid artery stenosis. PCSK6 has been associated with tumor invasiveness and extracellular matrix modulation in cancer but its function in the vasculature remains elusive. We demonstrate that PCSK6 deletion increases outward remodeling, reduces SMC differentiation and influences contractility in a murine model of flow-mediated remodeling. These results indicate that PCSK6 could be a potential target to reduce the risk of constrictive remodeling and restenosis

Lack of PCSK6 Increases Flow-Mediated Outward Arterial Remodeling in Mice

Proprotein convertases (PCSKs) process matrix metalloproteases and cytokines, but their function in the vasculature is largely unknown. Previously, we demonstrated upregulation of PCSK6 in atherosclerotic plaques from symptomatic patients, localization to smooth muscle cells (SMCs) in the fibrous cap and positive correlations with inflammation, extracellular matrix remodeling and cytokines. Here, we hypothesize that PCSK6 could be involved in flow-mediated vascular remodeling and aim to evaluate its role in the physiology of this process using knockout mice. Pcsk6-/- and wild type mice were randomized into control and increased blood flow groups and induced in the right common carotid artery (CCA) by ligation of the left CCA. The animals underwent repeated ultrasound biomicroscopy (UBM) examinations followed by euthanization with subsequent evaluation using wire myography, transmission electron microscopy or histology. The Pcsk6-/- mice displayed a flow-mediated increase in lumen circumference over time, assessed with UBM. Wire myography revealed differences in the flow-mediated remodeling response detected as an increase in lumen circumference at optimal stretch with concomitant reduction in active tension. Furthermore, a flow-mediated reduction in expression of SMC contractile markers SMA, MYH11 and LMOD1 was seen in the Pcsk6-/- media. Absence of PCSK6 increases outward remodeling and reduces medial contractility in response to increased blood flow

Transcriptomic and physiological analyses reveal temporal changes contributing to the delayed healing response to arterial injury in diabetic rats

Objective: Atherosclerosis is a leading cause of mortality in the rapidly growing population with diabetes mellitus. Vascular interventions in patients with diabetes can lead to complications attributed to defective vascular remodeling and impaired healing response in the vessel wall. In this study, we aim to elucidate the molecular differences in the vascular healing response over time using a rat model of arterial injury applied to healthy and diabetic conditions. Methods: Wistar (healthy) and Goto-Kakizaki (GK, diabetic) rats (n = 40 per strain) were subjected to left common carotid artery (CCA) balloon injury and euthanized at different timepoints: 0 and 20 hours, 5 days, and 2, 4, and 6 weeks. Noninvasive morphological and physiological assessment of the CCA was performed with ultrasound biomicroscopy (Vevo 2100) and corroborated with histology. Total RNA was isolated from the injured CCA at each timepoint, and microarray profiling was performed (n = 3 rats per timepoint; RaGene-1_0-st-v1 platform). Bioinformatic analyses were conducted using R software, DAVID bioinformatic tool, online STRING database, and Cytoscape software. Results: Significant increase in the neointimal thickness (P < .01; two-way analysis of variance) as well as exaggerated negative remodeling was observed after 2 weeks of injury in GK rats compared with heathy rats, which was confirmed by histological analyses. Bioinformatic analyses showed defective expression patterns for smooth muscle cells and immune cell markers, along with reduced expression of key extracellular matrix-related genes and increased expression of pro-thrombotic genes, indicating potential faults on cell regulation level. Transcription factor–protein-protein interaction analysis provided mechanistic evidence with an array of transcription factors dysregulated in diabetic rats. Conclusions: In this study, we have demonstrated that diabetic rats exhibit impaired arterial remodeling characterized by a delayed healing response. We show that increased contractile smooth muscle cell marker expression coincided with decreased matrix metalloproteinase expression, indicating a potential mechanism for a lack of extracellular matrix reorganization in the impaired vascular healing in GK rats. These results further corroborate the higher prevalence of restenosis in patients with diabetes and provide vital molecular insights into the mechanisms contributing to the impaired arterial healing response in diabetes. Moreover, the presented study provides the research community with the valuable longitudinal gene expression data bank for further exploration of diabetic vasculopathy. : Clinical Relevance: Vascular interventions causes injury to the arterial wall, which in turn induces a healing response to restore vessel wall homeostasis. However, in patients with diabetes, such interventions lead to exaggerated healing response and defective remodeling. There is a need to understand the molecular mechanisms underlying the defective healing response in diabetes. In this study, ultrasound biomicroscopy, histology, and microarray profiling were used to demonstrate the transcriptional and physiological changes at various timepoints following arterial injury in healthy Wistar and diabetic GK rats. This study also provides a database of longitudinal transcriptional changes for the research community to study vascular healing in diabetes

Log-ratio of silica to aluminium counts (ln(Si/Al)) from ODP site 108-658

Growing evidence suggests that the low atmospheric CO2 concentration of the ice ages resulted from enhanced storage of CO2 in the ocean interior, largely as a result of changes in the Southern Ocean1. Early in the most recent deglaciation, a reduction in North Atlantic overturning circulation seems to have driven CO2 release from the Southern Ocean**2, 3, 4, 5, but the mechanism connecting the North Atlantic and the Southern Ocean remains unclear. Biogenic opal export in the low-latitude ocean relies on silicate from the underlying thermocline, the concentration of which is affected by the circulation of the ocean interior. Here we report a record of biogenic opal export from a coastal upwelling system off the coast of northwest Africa that shows pronounced opal maxima during each glacial termination over the past 550,000 years. These opal peaks are consistent with a strong deglacial reduction in the formation of silicate-poor glacial North Atlantic intermediate water**2 (GNAIW). The loss of GNAIW allowed mixing with underlying silicate-rich deep water to increase the silicate supply to the surface ocean. An increase in westerly-wind-driven upwelling in the Southern Ocean in response to the North Atlantic change has been proposed to drive the deglacial rise in atmospheric CO2 (refs 3, 4). However, such a circulation change would have accelerated the formation of Antarctic intermediate water and sub-Antarctic mode water, which today have as little silicate as North Atlantic Deep Water and would have thus maintained low silicate concentrations in the Atlantic thermocline. The deglacial opal maxima reported here suggest an alternative mechanism for the deglacial CO2 release**5, 6. Just as the reduction in GNAIW led to upward silicate transport, it should also have allowed the downward mixing of warm, low-density surface water to reach into the deep ocean. The resulting decrease in the density of the deep Atlantic relative to the Southern Ocean surface promoted Antarctic overturning, which released CO2 to the atmosphere