Increased frequency of proangiogenic tunica intima endothelial kinase 2 (Tie2) expressing monocytes in individuals with type 2 diabetes mellitus.

BACKGROUND: Individuals with type 2 diabetes mellitus (T2DM) have an increased risk for developing macrovascular disease (MVD) manifested by atherosclerosis. Phenotypically and functionally different monocyte subsets (classical; CD14++CD16-, non-classical; CD14+CD16++, and intermediate; CD14++CD16+) including pro-angiogenic monocytes expressing Tie2 (TEMs) can be identified. Here we investigated monocyte heterogeneity and its association with T2DM and MVD. METHODS: Individuals with (N = 51) and without (N = 56) T2DM were recruited and allocated to "non-MVD" or "with MVD" (i.e., peripheral or coronary artery disease) subgroups. Blood monocyte subsets were quantified based on CD14, CD16 and Tie2 expression levels. Plasma levels of Tie2-ligands angiopoietin-1 and angiopoietin-2 were determined using ELISA. Carotid endarterectomy samples from individuals with (N = 24) and without (N = 22) T2DM were stained for intraplaque CD68+ macrophages (inflammation) and CD34+ (angiogenesis), as plaque vulnerability markers. RESULTS: Monocyte counts were similar between individuals with T2DM and healthy controls (non-diabetic, non-MVD). Non-classical monocytes were reduced (p < 0.05) in T2DM, whereas the percentage of TEMs within the intermediate subset was increased (p < 0.05). T2DM was associated with increased angiopoietin-1 (p < 0.05) and angiopoietin-2 (p = 0.0001) levels. Angiopoietin-2 levels were higher in T2DM individuals with MVD compared with non-MVD (p < 0.01). Endarterectomized plaques showed no differences in macrophage influx and microvessel number between individuals with and without T2DM. CONCLUSIONS: Monocyte subset distribution is altered in T2DM with reduced non-classical monocytes and increased TEM percentage in the intermediate monocyte subset. Increased angiopoietin-2 levels together with increased frequency of TEMs might promote plaque vulnerability in T2DM which could however not be confirmed at tissue level in advanced atherosclerotic lesions.

[18F]FDG and [18F]NaF as PET markers of systemic atherosclerosis progression: A longitudinal descriptive imaging study in patients with type 2 diabetes mellitus.

BACKGROUND: While [18F]-fluordeoxyglucose ([18F]FDG) uptake is associated with arterial inflammation, [18F]-sodium fluoride ([18F]NaF) is a marker for arterial micro-calcification. We aimed to investigate the prospective correlation between both PET markers over time and whether they are prospectively ([18F]FDG) and retrospectively ([18F]NaF) related to progression of systemic arterial disease in a longitudinal study in patients with type 2 diabetes mellitus (T2DM). METHODS: Baseline [18F]FDG PET/Low Dose (LD) Computed Tomography (CT) scans of ten patients with early T2DM without cardiovascular history (70% men, median age 63 years) were compared with five-year follow-up [18F]NaF/LDCT scans. Systemic activity was expressed as mean target-to-background ratio (meanTBR) by dividing the maximal standardized uptake value (SUVmax) of ten arteries by SUVmean of the caval vein. CT-assessed macro-calcifications were scored visually and expressed as calcified plaque (CP) score. Arterial stiffness was assessed with carotid-femoral pulse wave velocity (PWV). Five-year changes were expressed absolutely with delta (Δ) and relatively with %change. RESULTS: Baseline meanTBR[18F]FDG was strongly correlated with five-year follow-up meanTBR[18F]NaF (r = 0.709, P = .022). meanTBR[18F]NaF correlated positively with ΔCPscore, CPscore at baseline, and follow-up (r = 0.845, P = .002 and r = 0.855, P = .002, respectively), but not with %change in CPscore and PWV. CONCLUSION: This proof-of-concept study demonstrated that systemic arterial inflammation is an important pathogenetic factor in systemic arterial micro-calcification development.

18F-sodium fluoride positron emission tomography assessed microcalcifications in culprit and non-culprit human carotid plaques.

BACKGROUND: 18F-NaF positron emission tomography (PET) targets microcalcifications. We compared in vitro microPET assessed 18F-NaF uptake between culprit and non-culprit human carotid plaques. Furthermore, we compared 18F-NaF uptake with calcification visualized on microcomputed tomography (microCT). METHODS: Carotid plaques from stroke patients undergoing surgery were incubated in 18F-NaF and scanned using a microPET and a microCT scan. The average PET assessed 18F-NaF uptake was expressed as percentage of the incubation dose per gram (%Inc/g). 18F-NaF PET volume of interest (VOI) was compared with CT calcification VOI. RESULTS: 23 carotid plaques (17 culprit, 6 non-culprit) were included. The average 18F-NaF uptake in culprit carotid plaques was comparable with the uptake in non-culprit carotid plaques (median 2.32 %Inc/g [IQR 1.98 to 2.81] vs. median 2.35 %Inc/g [IQR 1.77 to 3.00], P = 0.916). Only a median of 10% (IQR 4 to 25) of CT calcification VOI showed increased 18F-NaF uptake, while merely a median of 35% (IQR 6 to 42) of 18F-NaF PET VOI showed calcification on CT. CONCLUSIONS: 18F-NaF PET represents a different stage in the calcification process than CT. We observed a similar PET assessed 18F-NaF uptake and pattern in culprit and non-culprit plaques of high-risk patients, indicating that this method may be of more value in early atherosclerotic stenosis development.

Angiotensin II induces reorganization of the actin cytoskeleton and myosin light-chain phosphorylation in podocytes through rho/ROCK-signaling pathway.

AIMS: In the present study, we have evaluated the effect of angiotensin II (Ang II) on actin cytoskeleton reorganization and myosin light-chain (MLC) phosphorylation in podocytes to demonstrate whether the Rho/Rho-associated coiled kinase (ROCK) pathway is involved podocyte injury. METHODS: Eighteen male Sprague-Dawley rats were divided into three groups and treated with Ang II, saline or telmisartan. Morphological changes were studied at 28 days after treatment. Immunohistochemistry and Western blotting were used to determine the renal expression of p-MLC and ROCK2. Cultured podocytes were treated with Ang II (10(-7 )M) with or without Rho-kinase inhibitor (Y27632, 10(-6 )M) for variable time periods. F-actin was visualized with fluorescein isothiocyanate (FITC)-conjugated phalloidin or tetraethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin. p-MLC expression was evaluated by immunofluorescence and Western blot. The activation of Rho/ROCK was evaluated by Western blot. RESULTS: The expression of p-MLC in glomeruli increased significantly in rats treated with Ang II when compared to the control rats as shown by Western blot (p < 0.05). In cultured podocytes, Rho A and ROCK2 increased after incubation with Ang II. Ang II increased the expression of ROCK2, which was accompanied with altered morphology, redistribution of actin and increased phosphorylation of MLC. The distribution of actin changed to a large extent, although overall quantitative differences were not observed. Addition of Y-27632 to podocytes treated with Ang II could ameliorate F-actin cytoskeleton remodeling and the increment in p-MLC expression. CONCLUSION: Ang II-induced podocyte cytoskeleton protein expression changing through the RhoA/ROCK2 p-MLC/F-actin pathway.

Complement mediated renal inflammation induced by donor brain death: role of renal C5a-C5aR interaction.

Kidneys retrieved from brain-dead donors have impaired allograft function after transplantation compared to kidneys from living donors. Donor brain death (BD) triggers inflammatory responses, including both systemic and local complement activation. The mechanism by which systemic activated complement contributes to allograft injury remains to be elucidated. The aim of this study was to investigate systemic C5a release after BD in human donors and direct effects of C5a on human renal tissue. C5a levels were measured in plasma from living and brain-dead donors. Renal C5aR gene and protein expression in living and brain-dead donors was investigated in renal pretransplantation biopsies. The direct effect of C5a on human renal tissue was investigated by stimulating human kidney slices with C5a using a newly developed precision-cut method. Elevated C5a levels were found in plasma from brain-dead donors in concert with induced C5aR expression in donor kidney biopsies. Exposure of precision-cut human kidney slices to C5a induced gene expression of pro-inflammatory cytokines IL-1 beta, IL-6 and IL-8. In conclusion, these findings suggest that systemic generation of C5a mediates renal inflammation in brain-dead donor grafts via tubular C5a-C5aR interaction. This study also introduces a novel in vitro technique to analyze renal cells in their biological environment.

Severely increased albuminuria in patients with type 2 diabetes mellitus is associated with increased subclinical atherosclerosis in femoral arteries with Na [18F]F activity as a proxy - The DETERMINE study.

BACKGROUND AND AIMS: Sodium [18F]fluoride (Na [18F]F) positron emission tomography imaging allows detailed visualization of early arterial micro-calcifications. This study aims to investigate atherosclerosis manifested by micro-calcification, macro-calcification, and aortic stiffness in patients with type 2 diabetes mellitus (T2DM) with and without albuminuria and severely decreased kidney function. METHODS: A cohort was stratified in four groups (N = 10 per group), based on KDIGO categories (G1-5 A1-3). G1-2A1 non-diabetic controls (median [IQR] estimated glomerular filtration rate (eGFR) in mL/min/1.73 m2 91 [81-104]), G1-2A1 with T2DM (eGFR 87 [84-93], and albumin-creatinin-ratio (ACR) in mg/mmol 0.35 [0.25-0.75]), G1-2A3 with T2DM (eGFR 85 [60-103], and ACR 74 [62-122], and G4A3 with T2DM (eGFR 19 [13-27] and ACR 131 [59-304]). RESULTS: Na [18F]F femoral artery grading score differed significantly in the groups with the highest Na [18F]F activity in A3 groups with T2DM (G1-2A3 with T2DM 228 [100-446] and G4A3 with T2DM 198 [113-578]) from the lowest groups of the G1-2A1 with T2DM (33 [0-93]) and in G1-2A1 non-diabetic controls (75 [0-200], p = 0.001). Aortic Na [18F]F activity and femoral artery computed tomography (CT)-assessed macro-calcification was increased in G4A3 with T2DM compared with G1-2A1 with T2DM (47.5 [33.8-73.8] vs. 17.5 [8.8-27.5] (p = 0.006) and 291 [170-511] vs. 12.2 [1.41-44.3] mg (p = 0.032), respectively). Carotid-femoral pulse wave velocity (PWV)-assessed aortic stiffness was significantly higher in both A3 groups with T2DM compared with G1-2A1 with T2DM (11.15 and 12.35 vs. 8.86 m/s, respectively (p = 0.009)). CONCLUSIONS: This study indicates that the presence of severely increased albuminuria in patients with T2DM is cross-sectionally associated with subclinical arterial disease in terms of micro-calcification and aortic stiffness. Additional decrease in kidney function was associated with advanced macro-calcifications.

Type 2 diabetes mellitus is associated with an imbalance in circulating endothelial and smooth muscle progenitor cell numbers.

AIMS/HYPOTHESIS: Individuals with type 2 diabetes mellitus have increased rates of macrovascular disease (MVD). Endothelial progenitor cells (EPCs), circulating angiogenic cells (CACs) and smooth muscle progenitor cells (SMPCs) are suggested to play a role in the pathogenesis of MVD. The relationship between vasoregenerative EPCs or CACs and damaging SMPCs and the development of accelerated MVD in diabetes is still unknown. We tried to elucidate whether EPC, CAC and SMPC numbers and differentiation capacities in vitro differ in patients with and without diabetes or MVD. METHODS: Peripheral blood was obtained from individuals with and without diabetes and MVD (coronary or peripheral artery disease). EPC and SMPC numbers were determined with flow cytometry. Furthermore, CAC and SMPC numbers were quantified after in vitro culture. Their in vitro differentiation capacity was investigated with real-time RT-PCR and quantitative immunofluorescence. RESULTS: In diabetic patients both EPC and CAC levels were reduced (1.3-fold [p < 0.05] and 1.5-fold [p < 0.05], respectively). CAC outgrowth from diabetic patients with MVD was reduced 1.5-fold compared with diabetic patients without MVD (p < 0.05). SMPC levels were similar between diabetic patients and healthy controls. The CAC/SMPC ratio of in vitro cultured progenitor cells was reduced 2.3-fold in samples from diabetic patients (p < 0.001). The differentiation capacity of CACs and SMPCs in vitro remained similar independently of diabetes or MVD. CONCLUSIONS/INTERPRETATION: The ratio between EPCs or CACs and SMPCs is disturbed in type 2 diabetes in favour of SMPCs. This may translate into reduced vascular repair capacity, thereby promoting MVD in type 2 diabetes.

Local medial microenvironment directs phenotypic modulation of smooth muscle cells after experimental renal transplantation.

Smooth muscle cells (SMCs) play a key role in the pathogenesis of occlusive vascular diseases, including transplant vasculopathy. Neointimal SMCs in experimental renal transplant vasculopathy are graft-derived. We propose that neointimal SMCs in renal allografts are derived from the vascular media resulting from a transplantation-induced phenotypic switch. We examined the molecular changes in the medial microenvironment that lead to phenotypic modulation of SMCs in rat renal allograft arteries with neointimal lesions. Dark Agouti donor kidneys were transplanted into Wistar Furth recipients and recovered at day 56. Neointimal and medial layers were isolated using laser microdissection. Gene expression was analyzed using low-density arrays and confirmed by immunostaining. In allografts, neointimal SMCs expressed increased levels of Tgf ß1 and Pdgfb. In medial allograft SMCs, gene expression of Ctgf, Tgf ß1 and Pdgfrb was upregulated. Gene expression of Klf4 was upregulated as well, while expression of Sm22α was downregulated. Finally, PDGF-BB-stimulated phenotypically modulated SMCs, as evidenced by reduced contractile function in vitro which was accompanied by increased Klf4 and Col1α1, and reduced α-Sma and Sm22α expression. In transplant vasculopathy, neointimal PDGF-BB induces phenotypic modulation of medial SMCs, through upregulation of KLF4 in the media to contribute to (further) expansion of the neointima.

Association of complement C3 gene variants with renal transplant outcome of deceased cardiac dead donor kidneys.

Local renal complement activation by the donor kidney plays an important role in the pathogenesis of renal injury inherent to kidney transplantation. Contradictory results were reported about the protective effects of the donor C3F allotype on renal allograft outcome. We investigated the influence of the donor C3F allotype on renal transplant outcome, taking all different donor types into account. C3 allotypes of 1265 donor-recipient pairs were determined and divided into four genotypic groups according to the C3F allotype of the donor and the recipient. The four genotypic groups were analyzed for association with primary nonfunction (PNF), delayed graft function, acute rejection, death-censored graft survival and patient survival. Considering all donor types, multivariable analysis found no association of the donor C3F allotype with renal allograft outcome. Also, for living and deceased brain-dead donors, no association with allograft outcome was found. Post hoc subgroup analysis within deceased cardiac dead (DCD) donors revealed an independent protective association of donor C3F allotype with PNF. This study shows that the donor C3F allotype is not associated with renal allograft outcome after kidney transplantation. Subgroup analysis within DCD donors revealed an independent protective association of the donor C3F allotype with PNF, which is preliminary and warrants further validation.

Perivascular adipose tissue-derived nitric oxide compensates endothelial dysfunction in aged pre-atherosclerotic apolipoprotein E-deficient rats.

BACKGROUND AND AIMS: Atherosclerosis is a major contributor to global mortality and is accompanied by vascular inflammation and endothelial dysfunction. Perivascular adipose tissue (PVAT) is an established regulator of vascular function with emerging implications in atherosclerosis. We investigated the modulation of aortic relaxation by PVAT in aged rats with apolipoprotein E deficiency (ApoE-/-) fed a high-fat diet as a model of early atherosclerosis. METHODS AND RESULTS: ApoE-/- rats (N = 7) and wild-type Sprague-Dawley controls (ApoE+/+, N = 8) received high-fat diet for 51 weeks. Hyperlipidemia was confirmed in ApoE-/- rats by elevated plasma cholesterol (p < 0.001) and triglyceride (p = 0.025) levels. Early atherosclerosis was supported by increased intima/media thickness ratio (p < 0.01) and ED1-positive macrophage influx in ApoE-/- aortic intima (p < 0.001). Inflammation in ApoE-/- PVAT was characteristic by an increased [18F]FDG uptake (p < 0.01), ED1-positive macrophage influx (p = 0.0003), mRNA expression levels of CD68 (p < 0.001) and IL-1ß (p < 0.01), and upregulated iNOS protein (p = 0.011). The mRNAs of MCP-1, IL-6 and adiponectin remained unchanged in PVAT. Aortic PVAT volume measured with micro-PET/CT was increased in ApoE-/- rats (p < 0.01). Maximal endothelium-dependent relaxation (EDR) to acetylcholine in ApoE-/- aortic rings without PVAT was severely impaired (p = 0.012) compared with controls, while ApoE-/- aortic rings with PVAT showed higher EDR than controls. All EDR responses were blocked by L-NMMA and the expression of eNOS mRNA was increased in ApoE-/- PVAT (p = 0.035). CONCLUSION: Using a rat ApoE-/- model of early atherosclerosis, we capture a novel mechanism by which inflammatory PVAT compensates severe endothelial dysfunction by contributing NO upon cholinergic stimulation.

Thiols as markers of redox status in type 1 diabetes mellitus.

INTRODUCTION: Type 1 diabetes mellitus (T1DM) is associated with inflammation and the production of reactive oxygen species (ROS). Systemically, free thiols (R-SH) can be oxidized by ROS and circulating R-SH concentrations may directly reflect the systemic redox status. In this study the association between R-SH and clinical parameters of T1DM, including glycated haemoglobin A1c (HbA1c), was investigated. This is of particular interest since thiols are amendable to therapeutic intervention. METHODS: As part of a prospective cohort study, data from 216 patients with a mean age of 45 (12) years, 57% male, diabetes duration 22 (16, 30) years and HbA1c of 60 (11) mmol/mol were examined. Baseline data were collected in 2002 and follow-up data in 2018. Cox proportional hazards regression analysis, with age, sex, HbA1c and R-SH, was used to assess prognostic factors for the development of complications. RESULTS: At baseline, the plasma concentration of R-SH was 281.8 ± 34.0 µM. In addition to a lower concentration of NT-proBNP in the highest R-SH quartile (305-379 µM) there were no differences in baseline characteristics between the quartiles of R-SH. The Pearson correlation coefficient for R-SH and NT-proBNP was -0.290 (p < 0.001). No significant correlation between R-SH and baseline HbA1c (r = -0.024, p = 0.726) was present. During follow-up, 42 macrovascular and 92 microvascular complications occurred. In Cox regression, R-SH was not a prognostic factor for the development of microvascular [hazard ratio (HR) 0.999 (95% confidence interval (CI) 0.993, 1.005)] and macrovascular [HR 0.993 (95% CI 0.984, 1.002)] complications. CONCLUSIONS: In addition to a negative association with NT-proBNP, no relevant relationships between R-SH and parameters of T1DM, including HbA1c, were present in this study.

Donor and recipient origin of mesenchymal and endothelial cells in chronic renal allograft remodeling.

Chronic transplant dysfunction (CTD) is the leading cause for limited kidney graft survival. Renal CTD is characterized by interstitial and vascular remodeling leading to interstitial fibrosis, tubular atrophy and transplant vasculopathy (TV). The origin of cells and pathogenesis of interstitial and vascular remodeling are still unknown. To study graft-versus-recipient origin of interstitial myofibroblasts, vascular smooth muscle cells (SMCs) and endothelial cells (ECs), we here describe a new rat model for renal CTD using Dark Agouti kidney donors and R26 human placental alkaline phosphatase transgenic Fischer344 recipients. This model showed the development of CTD within 12 weeks after transplantation. In interstitial remodeling, both graft- and recipient-derived cells contributed to a similar extent to the accumulation of myofibroblasts. In arteries with TV, we observed graft origin of neointimal SMCs and ECs, whereas in peritubular and glomerular capillaries, we detected recipient EC chimerism. These data indicate that, within the interstitial and vascular compartments of the transplanted kidney, myofibroblasts, SMCs and ECs involved in chronic remodeling are derived from different sources and suggest distinct pathogenetic mechanisms within the renal compartments.

Prolonged exclusive breastfeeding reduces autoimmune diabetes incidence and increases regulatory T-cell frequency in bio-breeding diabetes-prone rats.

BACKGROUND: Previously, we reported that exclusive breastfeeding delayed and partially protected bio-breeding diabetes-prone (BBDP) rats from spontaneous autoimmune diabetes development. To investigate whether this protection results from modulation of the (mucosal) immune system, the present study was designed to analyse the effect of nutrition early in life on the immune status of BBDP rats. METHODS: The breastfeeding period of BBDP pups was extended or not, while allowing half of the pups to eat during that period whereas the other half received only breast milk. Cytokine profiles as well as naturally occurring regulatory T-cell frequencies were measured over time in the mesenteric lymph nodes (MLNs) and spleen. RESULTS: Prolonged exclusive breastfeeding partially protects against autoimmune diabetes development and resulted in elevated levels of natural regulatory T cells (CD4(+) CD25(+) FoxP3(+)) in MLNs and spleen directly after weaning and throughout life. Stimulation of MLN cells from rats that ingested solid food during the nursing period showed massive secretion of interferon gamma (IFN-gamma), interleukin (IL)-4 and IL-10, whereas MLN cells from exclusive breastfed rats did not. In contrast, transforming growth factor beta (TGF-ss) was secreted equally by all groups. CONCLUSIONS: Prolonged exclusive breastfeeding partially protects BBDP rats from autoimmune diabetes development. Interestingly, ingestion of solid food during the weaning period completely abolishes this protective effect. The protective effect of exclusive breastfeeding correlates with higher levels of naturally occurring regulatory T cells throughout life and low cytokine secretion at weaning.

Clinical relevance of advanced glycation endproducts for vascular surgery.

Atherosclerosis is the main contributor to cardiovascular disease and leads to intimal plaque formation, which may progress to plaque rupture with subsequent thromboembolic events and/or occlusion of the arterial lumen. There is increasing evidence that the development or progression of atherosclerosis is associated with advanced glycation endproducts (AGEs). AGEs are a heterogeneous group of compounds formed by the non-enzymatic reaction of reducing sugars with proteins, lipids, and nucleic acids. An increased understanding of the mechanisms of formation and interaction of AGEs has allowed the development of several potential anti-AGE strategies. This review summarizes AGE formation and biochemistry, the pathogeneic role of AGEs in cardiovascular disease, anti-AGE therapies and clinical relevance to vascular surgery.

Origin of neointimal endothelium and alpha-actin-positive smooth muscle cells in transplant arteriosclerosis.

The development of transplant arteriosclerosis (TA) is today's most important problem in clinical organ transplantation. Histologically, TA is characterized by perivascular inflammation and progressive intimal thickening. Current thought on this process of vascular remodeling assumes that neointimal vascular smooth muscle (VSM) cells and endothelium in TA are graft-derived, holding that medial VSM cells proliferate and migrate into the subendothelial space in response to signals from inflammatory cells and damaged graft endothelium. Using MHC class I haplotype-specific immunohistochemical staining and single-cell PCR analyses, we show that the neointimal alpha-actin-positive VSM cells in rat aortic or cardiac allografts are of recipient and not of donor origin. In aortic but not in cardiac allografts, recipient-derived endothelial cells (ECs) replaced donor endothelium. Cyclosporine treatment prevents neointima formation and preserves the vascular media in aortic allografts. Recipient-derived ECs do not replace graft endothelium after cyclosporine treatment. We propose that, although it progresses beyond the needs of functional repair, TA reflects the activity of a normal healing process that restores vascular wall function following allograft-induced immunological injury.

Diverging effects of diabetes mellitus in patients with peripheral artery disease and abdominal aortic aneurysm and the role of advanced glycation end-products: ARTERY study - protocol for a multicentre cross-sectional study.

INTRODUCTION: Diabetes mellitus is a well-defined risk factor for peripheral artery disease (PAD), but protects against the development and growth of abdominal aortic aneurysm (AAA). Diabetes mellitus is associated with arterial stiffening and peripheral arterial media sclerosis. Advanced glycation end-products (AGEs) are increased in diabetes mellitus and cardiovascular disease. AGEs are known to form cross-links between proteins and are associated with arterial stiffness. Whether AGEs contribute to the protective effects of diabetes mellitus in AAA is unknown. Therefore, the ARTERY (Advanced glycation end-pRoducts in patients with peripheral arTery disEase and abdominal aoRtic aneurYsm) study is designed to evaluate the role of AGEs in the diverging effects of diabetes mellitus on AAA and PAD. METHODS AND ANALYSIS: This cross-sectional multicentre study will compare the amount, type and location of AGEs in the arterial wall in a total of 120 patients with AAA or PAD with and without diabetes mellitus (n=30 per subgroup). Also, local and systemic vascular parameters, including pulse wave velocity, will be measured to evaluate the association between arterial stiffness and AGEs. Finally, AGEs will be measured in serum, urine, and assessed in skin with skin autofluorescence using the AGE Reader. ETHICS AND DISSEMINATION: This study is approved by the Medical Ethics committees of University Medical Center Groningen, Martini Hospital and Medisch Spectrum Twente, the Netherlands. Study results will be disseminated through peer-reviewed journals and scientific events. TRIAL REGISTRATION NUMBER: trialregister.nl NTR 5363.

Polymeric microspheres for the sustained release of a protein-based drug carrier targeting the PDGFß-receptor in the fibrotic kidney.

Injectable sustained release drug delivery systems are an attractive alternative for the intravenous delivery of therapeutic proteins. In particular, for chronic diseases such as fibrosis, this approach could improve therapy by reducing the administration frequency while avoiding large variations in plasma levels. In fibrotic tissues the platelet-derived growth factor receptor beta (PDGFßR) is highly upregulated, which provides a target for site-specific delivery of drugs. Our aim was to develop an injectable sustained release formulation for the subcutaneous delivery of the PDGFßR-targeted drug carrier protein pPB-HSA, which is composed of multiple PDGFßR-recognizing moieties (pPB) attached to human serum albumin (HSA). We used blends of biodegradable multi-block copolymers with different swelling degree to optimize the release rate using the model protein HSA from microspheres produced via a water-in-oil-in-water double emulsion evaporation process. The optimized formulation containing pPB-HSA, showed complete release in vitro within 14days. After subcutaneous administration to mice suffering from renal fibrosis pPB-HSA was released from the microspheres and distributed into plasma for at least 7days after administration. Furthermore, we demonstrated an enhanced accumulation of pPB-HSA in the fibrotic kidney. Altogether, we show that subcutaneously administered polymeric microspheres present a suitable sustained release drug delivery system for the controlled systemic delivery for proteins such as pPB-HSA.

Cystathionine γ-lyase is expressed in human atherosclerotic plaque microvessels and is involved in micro-angiogenesis.

Atherosclerotic plaques are classically divided into stable and vulnerable plaques. Vulnerable plaques are prone to rupture with a risk for infarction. High intraplaque microvessel density predisposes to plaque vulnerability. Hydrogen sulfide (H2S) is a proangiogenic gasotransmitter which is endogenously produced by cystathionine γ-lyase (CSE), and is believed to have vasculoprotective effects. However, due to its proangiogenic effects, H2S may result in pathological angiogenesis in atherosclerotic plaques, thereby increasing plaque vulnerability. The aim of this study was to determine CSE expression pattern in atherosclerotic plaques, and investigate whether CSE is involved in micro-angiogenesis in vitro. Endarterectomy plaques were studied for CSE expression, and the role of CSE in micro-angiogenesis was studied in vitro. CSE is expressed in plaques with similar levels in both stable and vulnerable plaques. CSE co-localized with von Willebrand Factor-positive microvessel endothelial cells and alpha-smooth-muscle actin-positive SMCs. In vitro, inhibition of CSE in HMEC-1 reduced tube formation, cell viability/proliferation, and migration which was restored after culture in the presence of H2S donor GYY4137. CSE is expressed in intraplaque microvessels, and H2S is a stimulator of micro-angiogenesis in vitro. Due to this pro-angiogenic effect, high levels of CSE in atherosclerotic plaques may be a potential risk for plaque vulnerability.

Chronic allograft rejection associated vasculopathy and synthetic biodegradable vascular grafts: a lesson to learn?

In chronic allografts, graft vessels eventually develop so-called "transplant vascular sclerosis" or "intimal hyperplasia". A major question is whether the cells in the neointima are donor or recipient derived. The process of transplant vascular sclerosis closely resembles the remodeling of the vascular wall as seen when synthetic biodegradable small caliber vascular grafts are implanted. In this model, the cells in the newly developing neointima as well as neomedia are, by definition, recipient derived. By using cardiac allografts as well as aortic allografts exchanged between a female donor and a male recipient (rats), the origin of the neointimal vascular smooth muscle cells could be traced by looking for the Y-chromosome in isolated (alpha-actin positive) intimal cells using PCR. In both models these intimal cells were found to be of recipient-origin. It is proposed, that, basically, this remodeling process is part of a normal healing process. Whereas in biodegradable grafts this "healing process" appears to be self limiting, in allografts the process goes on beyond the needs of functional repair, eventually, in some cases, leading to total vascular occlusion. Future therapeutic protocols might try and aim at controlling this essentially normal repair process.

Exogenous administration of thiosulfate, a donor of hydrogen sulfide, attenuates angiotensin II-induced hypertensive heart disease in rats.

BACKGROUND AND PURPOSE: Hypertension is an important mediator of cardiac damage and remodelling. Hydrogen sulfide (H2S) is an endogenously produced gasotransmitter with cardioprotective properties. However, it is not yet in clinical use. We, therefore, investigated the protective effects of sodium thiosulfate (STS), a clinically applicable H2 S donor substance, in angiotensin II (Ang II)-induced hypertensive cardiac disease in rats. EXPERIMENTAL APPROACH: Male Sprague Dawley rats were infused with Ang II (435 ng kg min(-1)) or saline (control) for 3 weeks via s.c. placed osmotic minipumps. During these 3 weeks, rats received i.p. injections of either STS, NaHS or vehicle (0.9% NaCl). KEY RESULTS: Compared with controls, Ang II infusion caused an increase in systolic and diastolic BP with associated cardiac damage as evidenced by cardiac hypertrophy, an increase in atrial natriuretic peptide (ANP) mRNA, cardiac fibrosis and increased oxidative stress. Treatment with NaHS and STS prevented the development of hypertension and the increase in ANP mRNA levels. Furthermore, the degree of cardiac hypertrophy, the extent of histological fibrosis in combination with the expression of profibrotic genes and the levels of oxidative stress were all significantly decreased. CONCLUSIONS AND IMPLICATIONS: Ang II-induced hypertensive cardiac disease can be attenuated by treatment with STS and NaHS. Although BP regulation is the most plausible mechanism of cardiac protection, the antifibrotic and antioxidant properties of released sulfide may also contribute to their effects. Our data show that H2 S might be a valuable addition to the already existing antihypertensive and cardioprotective therapies.