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Background Arteriovenous fistula (AVF) maturation failure is a main limitation of vascular access. Maturation is determined by the intricate balance between outward remodeling and intimal hyperplasia, whereby endothelial cell dysfunction, platelet aggregation, and vascular smooth muscle cell (VSMC) proliferation play a crucial role. von Willebrand Factor (vWF) is an endothelial cell–derived protein involved in platelet aggregation and VSMC proliferation. We investigated AVF vascular remodeling in vWF‐deficient mice and vWF expression in failed and matured human AVFs. Methods and Results Jugular‐carotid AVFs were created in wild‐type and vWF−/− mice. AVF flow was determined longitudinally using ultrasonography, whereupon AVFs were harvested 14 days after surgery. VSMCs were isolated from vena cavae to study the effect of vWF on VSMC proliferation. Patient‐matched samples of the basilic vein were obtained before brachio‐basilic AVF construction and during superficialization or salvage procedure 6 weeks after AVF creation. vWF deficiency reduced VSMC proliferation and macrophage infiltration in the intimal hyperplasia. vWF−/− mice showed reduced outward remodeling (1.5‐fold, P=0.002) and intimal hyperplasia (10.2‐fold, P

Post-transcriptional control of RNA is important in health and disease. Here, the authors show that the RNA-binding protein Quaking guides pre-mRNA splicing and transcript abundance during monocyte to macrophage differentiation, and that Quaking depletion impairs pro-atherogenic foam cell formation.

Atherosclerosis is the underlying pathology in a major part of cardiovascular disease, the leading cause of mortality in developed countries. The infiltration of monocytes into the vessel walls of large arteries is a key denominator of atherogenesis, making monocytes accountable for the development of atherosclerosis. With the development of high-throughput transcriptome profiling platforms and cytometric methods for circulating cells, it is now feasible to study in-depth the predicted functional change of circulating monocytes reflected by changes of gene expression in certain pathways and correlate the changes to disease outcome. Neuroimmune guidance cues comprise a group of circulating- and cell membrane-associated signaling proteins that are progressively involved in monocyte functions. Here, we employed the CIRCULATING CELLS study cohort to classify cardiovascular disease patients and healthy individuals in relation to their expression of neuroimmune guidance cues in circulating monocytes. To cope with the complexity of human datasets featured by noisy data, nonlinearity and multidimensionality, we assessed various machine-learning methods. Of these, the linear discriminant analysis, Naïve Bayesian model and stochastic gradient boost model yielded perfect or near-perfect sensibility and specificity and revealed that expression levels of the neuroimmune guidance cues SEMA6B, SEMA6D and EPHA2 in circulating monocytes were of predictive values for cardiovascular disease outcome.

We recently reported that loss of hyaluronan (HA) from the endothelial glycocalyx leads to loss of vessel stability in specific microcirculatory vascular beds. Here we hypothesized that such derangements in the glycocalyx may also impair the adaptive response to vascular ischemia. Endothelial specific conditional hyaluronan synthase 2-KO (Has2-cKO) mice revealed reduced endothelial HA expression and lower hindlimb perfusion at baseline compared to control mice. After a single ligation of the common femoral artery in these mice, we observed dysregulated angiogenesis in the gastrocnemius muscle which did not restore capillary perfusion. Mechanistically, decreased endothelial binding of the pericyte-derived molecule angiopoietin1 (Ang1) could be observed in the Has2-cKO mouse. In vitro angiogenesis assays with an endothelial cell-pericyte coculture confirmed such disturbed Ang1-TIE2 signaling resulting in excessive angiogenesis upon loss of HA. These data could be of relevance to diabetes patients, where we confirm loss of endothelial HA in the microcirculation of muscle tissue, indicating that this may contribute to the known disturbed adaptation to ischemia in these patients. In summary, loss of endothelial HA results in impaired microvascular perfusion and endothelial stability in ischemic gastrocnemius muscle. Endothelial HA is a potential target to improve angiogenic therapy in diabetic patients with critical limb ischemia.

In the pathophysiologic setting of acute and chronic kidney injury, the excessive activation and recruitment of blood-borne monocytes prompts their differentiation into inflammatory macrophages, a process that leads to progressive glomerulosclerosis and interstitial fibrosis. Importantly, this differentiation of monocytes into macrophages requires the meticulous coordination of gene expression at both the transcriptional and post-transcriptional level. The transcriptomes of these cells are ultimately determined by RNA-binding proteins such as QUAKING (QKI), that define their pre-mRNA splicing and mRNA transcript patterns. Using two mouse models, namely (1) quaking viable mice (qkv) and (2) the conditional deletion in the myeloid cell lineage using the lysozyme 2-Cre (QKIFL/FL;LysM-Cre mice), we demonstrate that the abrogation of QKI expression in the myeloid cell lineage reduces macrophage infiltration following kidney injury induced by unilateral urethral obstruction (UUO). The qkv and QKIFL/FL;LysM-Cre mice both showed significant diminished interstitial collagen deposition and fibrosis in the UUO-damaged kidney, as compared to wild-type littermates. We show that macrophages isolated from QKIFL/FL;LysM-Cre mice are associated with defects in pre-mRNA splicing. Our findings demonstrate that reduced expression of the alternative splice regulator QKI in the cells of myeloid lineage attenuates renal interstitial fibrosis, suggesting that inhibition of this splice regulator may be of therapeutic value for certain kidney diseases.

COPD is a pulmonary disorder often accompanied by cardiovascular disease (CVD), and current treatment of this comorbidity is suboptimal. Systemic inflammation in COPD triggered by smoke and microbial exposure is suggested to link COPD and CVD. Mesenchymal stromal cells (MSC) possess anti-inflammatory capacities and MSC treatment is considered an attractive treatment option for various chronic inflammatory diseases. Therefore, we investigated the immunomodulatory properties of MSC in an acute and chronic model of lipopolysaccharide (LPS)-induced inflammation, emphysema and atherosclerosis development in APOE*3-Leiden (E3L) mice.Hyperlipidemic E3L mice were intranasally instilled with 10 μg LPS or vehicle twice in an acute 4-day study, or twice weekly during 20 weeks Western-type diet feeding in a chronic study. Mice received 0.5x106 MSC or vehicle intravenously twice after the first LPS instillation (acute study) or in week 14, 16, 18 and 20 (chronic study). Inflammatory parameters were measured in bronchoalveolar lavage (BAL) and lung tissue. Emphysema, pulmonary inflammation and atherosclerosis were assessed in the chronic study.In the acute study, intranasal LPS administration induced a marked systemic IL-6 response on day 3, which was inhibited after MSC treatment. Furthermore, MSC treatment reduced LPS-induced total cell count in BAL due to reduced neutrophil numbers. In the chronic study, LPS increased emphysema but did not aggravate atherosclerosis. Emphysema and atherosclerosis development were unaffected after MSC treatment.These data show that MSC inhibit LPS-induced pulmonary and systemic inflammation in the acute study, whereas MSC treatment had no effect on inflammation, emphysema and atherosclerosis development in the chronic study.

BackgroundHypercholesterolemia is a major risk factor for ischemic heart disease including acute myocardial infarction. However, long-term effects of hypercholesterolemia in a rodent myocardial ischemia-reperfusion injury model are unknown. Therefore, the effects of diet-induced hypercholesterolemia on cardiac function and remodeling were investigated up to eight weeks after myocardial ischemia-reperfusion (MI-R) injury which was induced in either normocholesterolemic (NC-MI) or hypercholesterolemic (HC-MI) APOE*3-Leiden mice.MethodsLeft ventricular (LV) dimensions were serially assessed using parasternal long-axis echocardiography followed by LV pressure-volume measurements. Subsequently, infarct size and the inflammatory response were analyzed by histology and fluorescence-activated cell sorting (FACS) analysis.ResultsIntrinsic LV function eight weeks after MI-R was significantly impaired in HC-MI compared to NC-MI mice as assessed by end-systolic pressure, dP/dtMAX, and -dP/dtMIN. Paradoxically, infarct size was significantly decreased in HC-MI compared to NC-MI mice, accompanied by an increased wall thickness. Hypercholesterolemia caused a pre-ischemic peripheral monocytosis, in particular of Ly-6Chi monocytes whereas accumulation of macrophages in the ischemic-reperfused myocardium of HC-MI mice was decreased.ConclusionDiet-induced hypercholesterolemia caused impaired LV function eight weeks after MI-R injury despite a reduced post-ischemic infarct size. This was preceded by a pre-ischemic peripheral monocytosis, while there was a suppressed accumulation of inflammatory cells in the ischemic-reperfused myocardium after eight weeks. This experimental model using hypercholesterolemic APOE*3-Leiden mice exposed to MI-R seems suitable to study novel cardioprotective therapies in a more clinically relevant animal model.

We recently demonstrated that the adult human kidney cortex contains a perivascular stromal cell (kPSC) that shows organotypic properties and is important for repair and stabilisation of kidney function. Not only the kidney cortex but also the kidney capsule contains stromal cells that are important for the three dimensional organisation of the kidney during nephrogenesis. They provide the barrier function of the capsule which is critical for homeostatic processes such as pressure natriuresis. We postulated that stromal cells derived from the kidney capsule may therefore also have specific properties and functions. To this end, we isolated these capsule mesenchymal stromal cells (cMSC) from human cadaveric kidneys that were not suitable for transplantation. There were several similarities between cMSCs and kPSCs including support of vascular plexus formation, phenotypic marker expression and resistance against myofibroblast transformation. However, compared to kPSCs, cMSCs showed distinct mRNA and miRNA expression profiles, showed increased immunosuppressive capacity, and displayed strongly reduced HGF production, contributing to the inability to enhance kidney epithelial repair. Therefore cMSCs are a distinct, novel human kidney-derived MSC-population and these data underpin the large functional diversity of phenotypic similar stromal cells in relation to their anatomic site, even within one organ.

AimsWe investigated the role of the TLR4-accessory molecule RP105 (CD180) in post-ischemic neovascularization, i.e. arteriogenesis and angiogenesis. TLR4-mediated activation of pro-inflammatory Ly6Chi monocytes is crucial for effective neovascularization. Immunohistochemical analyses revealed that RP105+ monocytes are present in the perivascular space of remodeling collateral arterioles. As RP105 inhibits TLR4 signaling, we hypothesized that RP105 deficiency would lead to an unrestrained TLR4-mediated inflammatory response and hence to enhanced blood flow recovery after ischemia.Methods and resultsRP105-/- and wild type (WT) mice were subjected to hind limb ischemia and blood flow recovery was followed by Laser Doppler Perfusion Imaging. Surprisingly, we found that blood flow recovery was severely impaired in RP105-/- mice. Immunohistochemistry showed that arteriogenesis was reduced in these mice compared to the WT. However, both in vivo and ex vivo analyses showed that circulatory pro-arteriogenic Ly6Chi monocytes were more readily activated in RP105-/- mice. FACS analyses showed that Ly6Chi monocytes became activated and migrated to the affected muscle tissues in WT mice following induction of hind limb ischemia. Although Ly6Chi monocytes were readily activated in RP105-/- mice, migration into the ischemic tissues was hampered and instead, Ly6Chi monocytes accumulated in their storage compartments, bone marrow and spleen, in RP105-/- mice.ConclusionsRP105 deficiency results in an unrestrained inflammatory response and monocyte over-activation, most likely due to the lack of TLR4 regulation. Inappropriate, premature systemic activation of pro-inflammatory Ly6Chi monocytes results in reduced infiltration of Ly6Chi monocytes in ischemic tissues and in impaired blood flow recovery.

BackgroundRP105 (CD180) is TLR4 homologue lacking the intracellular TLR4 signaling domain and acts a TLR accessory molecule and physiological inhibitor of TLR4-signaling. The role of RP105 in vascular remodeling, in particular post-interventional remodeling is unknown.Methods and resultsTLR4 and RP105 are expressed on vascular smooth muscle cells (VSMC) as well as in the media of murine femoral artery segments as detected by qPCR and immunohistochemistry. Furthermore, the response to the TLR4 ligand LPS was stronger in VSMC from RP105(-/-) mice resulting in a higher proliferation rate. In RP105(-/-) mice femoral artery cuff placement resulted in an increase in neointima formation as compared to WT mice (4982 ± 974 µm(2) vs.1947 ± 278 µm(2),p = 0.0014). Local LPS application augmented neointima formation in both groups, but in RP105(-/-) mice this effect was more pronounced (10316±1243 µm(2) vs.4208 ± 555 µm(2),p = 0.0002), suggesting a functional role for RP105. For additional functional studies, the extracellular domain of murine RP105 was expressed with or without its adaptor protein MD1 and purified. SEC-MALSanalysis showed a functional 2∶2 homodimer formation of the RP105-MD1 complex. This protein complex was able to block the TLR4 response in whole blood ex-vivo. In vivo gene transfer of plasmid vectors encoding the extracellular part of RP105 and its adaptor protein MD1 were performed to initiate a stable endogenous soluble protein production. Expression of soluble RP105-MD1 resulted in a significant reduction in neointima formation in hypercholesterolemic mice (2500 ± 573 vs.6581 ± 1894 µm(2),pConclusionRP105 is a potent inhibitor of post-interventional neointima formation.

AIMS: In collateral development (i.e. arteriogenesis), mononuclear cells are important and exist as a heterogeneous population consisting of pro-inflammatory and anti-inflammatory/repair-associated cells. Protease-activated receptor (PAR)1 and PAR2 are G-protein-coupled receptors that are both expressed by mononuclear cells and are involved in pro-inflammatory reactions, while PAR2 also plays a role in repair-associated responses. Here, we investigated the physiological role of PAR1 and PAR2 in arteriogenesis in a murine hind limb ischemia model. METHODS AND RESULTS: PAR1-deficient (PAR1-/-), PAR2-deficient (PAR2-/-) and wild-type (WT) mice underwent femoral artery ligation. Laser Doppler measurements revealed reduced post-ischemic blood flow recovery in PAR2-/- hind limbs when compared to WT, while PAR1-/- mice were not affected. Upon ischemia, reduced numbers of smooth muscle actin (SMA)-positive collaterals and CD31-positive capillaries were found in PAR2-/- mice when compared to WT mice, whereas these parameters in PAR1-/- mice did not differ from WT mice. The pool of circulating repair-associated (Ly6C-low) monocytes and the number of repair-associated (CD206-positive) macrophages surrounding collaterals in the hind limbs were increased in WT and PAR1-/- mice, but unaffected in PAR2-/- mice. The number of repair-associated macrophages in PAR2-/- hind limbs correlated with CD11b- and CD115-expression on the circulating monocytes in these animals, suggesting that monocyte extravasation and M-CSF-dependent differentiation into repair-associated cells are hampered. CONCLUSION: PAR2, but not PAR1, is involved in arteriogenesis and promotes the repair-associated response in ischemic tissues. Therefore, PAR2 potentially forms a new pro-arteriogenic target in coronary artery disease (CAD) patients.