Paracrine effects of mesenchymal stem cells enhance vascular regeneration in ischemic murine skin

New theories on the regeneration of ischemic vasculature have emerged indicating a pivotal role of adult stem cells. The aim of this study was to investigate homing and hemodynamic effects of circulating bone marrow-derived mesenchymal stem cells (MSCs) in a critically ischemic murine skin flap model. Bone marrow-derived mesenchymal stem cells (Lin(-)CD105(+)) were harvested from GFP(+)-donor mice and transferred to wildtype C57BL/6 mice. Animals receiving GFP(+)-fibroblasts served as a control group. Laser scanning confocal microscopy and intravital fluorescence microscopy were used for morphological analysis, monitoring and quantitative assessment of the stem cell homing and microhemodynamics over two weeks. Immunohistochemical staining was performed for GFP, eNOS, iNOS, VEGF. Tissue viability was analyzed by TUNEL-assay. We were able to visualize perivascular homing of MSCs in vivo. After 4 days, MSCs aligned along the vascular wall without undergoing endothelial or smooth muscle cell differentiation during the observation period. The gradual increase in arterial vascular resistance observed in the control group was abolished after MSC administration (P<0.01). At capillary level, a strong angiogenic response was found from day 7 onwards. Functional capillary density was raised in the MSC group to 197% compared to 132% in the control group (P<0.01). Paracrine expression of VEGF and iNOS, but not eNOS could be shown in the MSC group but not in the controls. In conclusion, we demonstrated that circulating bone marrow-derived MSCs home to perivascular sites in critically ischemic tissue, exhibits paracrine function and augment microhemodynamics. These effects were mediated through arteriogenesis and angiogenesis, which contributed to vascular regeneration.

Lowered microvascular vessel wall oxygen consumption augments tissue pO2 during PgE1-induced vasodilation

Continuous infusion of intravenous prostaglandin E1 (PgE1, 2.5 mug/kg/min) was used to determine how vasodilation affects oxygen consumption of the microvascular wall and tissue pO(2) in the hamster window chamber model. While systemic measurements (mean arterial pressure and heart rate) and central blood gas measurements were not affected, PgE1 treatment caused arteriolar (64.6 +/- 25.1 microm) and venular diameter (71.9 +/- 29.5 microm) to rise to 1.15 +/- 0.21 and 1.06 +/- 0.19, respectively, relative to baseline. Arteriolar (3.2 x 10(-2) +/- 4.3 x 10(-2) nl/s) and venular flow (7.8 x 10(-3) +/- 1.1 x 10(-2)/s) increased to 1.65 +/- 0.93 and 1.32 +/- 0.72 relative to baseline. Interstitial tissue pO(2) was increased significantly from baseline (21 +/- 8 to 28 +/- 7 mmHg; P < 0.001). The arteriolar vessel wall gradient, a measure of oxygen consumption by the microvascular wall decreased from 20 +/- 6 to 16 +/- 3 mmHg (P < 0.001). The arteriolar vessel wall gradient, a measure of oxygen consumption by the vascular wall, decreased from 20 +/- 6 to 16 +/- 3 mmHg (P < 0.001). This reduction reflects a 20% decrease in oxygen consumption by the vessel wall and up to 50% when cylindrical geometry is considered. The venular vessel wall gradient decreased from 12 +/- 4 to 9 +/- 4 mmHg (P < 0.001). Thus PgE1-mediated vasodilation has a positive microvascular effect: enhancement of tissue perfusion by increasing flow and then augmentation of tissue oxygenation by reducing oxygen consumption by the microvascular wall.

Alpha4beta1 integrin mediates the recruitment of immature dendritic cells across the blood-brain barrier during experimental autoimmune encephalomyelitis

Dendritic cells (DCs) within the CNS are recognized to play an important role in the effector phase and propagation of the immune response in experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. However, the mechanisms regulating DC trafficking into the CNS still need to be characterized. In this study, we show by performing intravital fluorescence videomicroscopy of the inflamed spinal cord white-matter microvasculature in SJL mice with EAE that immature, and to a lesser extent, LPS-matured, bone marrow-derived DCs efficiently interact with the CNS endothelium by rolling, capturing, and firm adhesion. Immature but not LPS-matured DCs efficiently migrated across the wall of inflamed parenchymal microvessels into the CNS. Blocking alpha4 integrins interfered with the adhesion but not the rolling or capturing of immature and LPS-matured DCs to the CNS microvascular endothelium, inhibiting their migration across the vascular wall. Functional absence of beta1 integrins but not of beta7 integrins or alpha4beta7 integrin similarly reduced the adhesion of immature DCs to the CNS microvascular endothelium, demonstrating that alpha4beta1 but not alpha4beta7 integrin mediates this step of immature DCs interaction with the inflamed blood-brain barrier during EAE. Our study shows that during EAE, especially immature DCs migrate into the CNS, where they may be crucial for the perpetuation of the CNS-targeted autoimmune response. Thus therapeutic targeting of alpha4 integrins affects DC trafficking into the CNS and may therefore lead to the resolution of the CNS autoimmune inflammation by reducing the number of CNS professional APCs.

Cardiovascular surgery in Marfan syndrome: implications of new molecular concepts in thoracic aortic disease

Acute dissection and rupture of aortic aneurysms comprise for 1-2% of all deaths in industrialized countries. Dilation of the aorta is caused by a multitude of mechanisms including inherited connective tissue disorders such as Marfan syndrome (MFS). MFS is one of the most common inherited connective tissue disorders affecting 1 in 5000 individuals. Although the phenotype of MFS can be quite variable, aneurysmal dilation of the aortic root and consecutive acute aortic dissection is the leading cause of death in this patient population. Over the past years it has been shown that a comprehensive understanding of this disorder provides greater understanding of vascular wall biology and identifies pathways relevant to aortic aneurysms and dissection in general. The current review discusses the surgical management of patients with MFS with a special emphasis on indications for surgery in this complex group of patients.

Molecular mechanisms involved in T cell migration across the blood-brain barrier

In the healthy individuum lymphocyte traffic into the central nervous system (CNS) is very low and tightly controlled by the highly specialized blood-brain barrier (BBB). In contrast, under inflammatory conditions of the CNS such as in multiple sclerosis or in its animal model experimental autoimmune encephalomyelitis (EAE) circulating lymphocytes and monocytes/macrophages readily cross the BBB and gain access to the CNS leading to edema, inflammation and demyelination. Interaction of circulating leukocytes with the endothelium of the blood-spinal cord and blood-brain barrier therefore is a critical step in the pathogenesis of inflammatory diseases of the CNS. Leukocyte/endothelial interactions are mediated by adhesion molecules and chemokines and their respective chemokine receptors. We have developed a novel spinal cord window preparation, which enables us to directly visualize CNS white matter microcirculation by intravital fluorescence videomicroscopy. Applying this technique of intravital fluorescence videomicroscopy we could provide direct in vivo evidence that encephalitogenic T cell blasts interact with the spinal cord white matter microvasculature without rolling and that alpha4-integrin mediates the G-protein independent capture and subsequently the G-protein dependent adhesion strengthening of T cell blasts to microvascular VCAM-1. LFA-1 was found to neither mediate the G-protein independent capture nor the G- protein dependent initial adhesion strengthening of encephalitogenic T cell blasts within spinal cord microvessel, but was rather involved in T cell extravasation across the vascular wall into the spinal cord parenchyme. Our observation that G-protein mediated signalling is required to promote adhesion strengthening of encephalitogenic T cells on BBB endothelium in vivo suggested the involvement of chemokines in this process. We found functional expression of the lymphoid chemokines CCL19/ELC and CCL21/SLC in CNS venules surrounded by inflammatory cells in brain and spinal cord sections of mice afflicted with EAE suggesting that the lymphoid chemokines CCL19 and CCL21 besides regulating lymphocyte homing to secondary lymphoid tissue might be involved in T lymphocyte migration into the immuneprivileged CNS during immunosurveillance and chronic inflammation. Here, I summarize our current knowledge on the sequence of traffic signals involved in T lymphocyte recruitment across the healthy and inflamed blood-brain and blood-spinal cord barrier based on our in vitro and in vivo investigations.

Perivascular spaces and the two steps to neuroinflammation

Immune cells enter the central nervous system (CNS) from the circulation under normal conditions for immunosurveillance and in inflammatory neurologic diseases. This review describes the distinct anatomic features of the CNS vasculature that permit it to maintain parenchymal homeostasis and which necessitate specific mechanisms for neuroinflammation to occur. We review the historical evolution of the concept of the blood-brain barrier and discuss distinctions between diffusion/transport of solutes and migration of cells from the blood to CNS parenchyma. The former is regulated at the level of capillaries, whereas the latter takes place in postcapillary venules. We summarize evidence that entry of immune cells into the CNS parenchyma in inflammatory conditions involves 2 differently regulated steps: transmigration of the vascular wall into the perivascular space and progression across the glia limitans into the parenchyma.

T-cadherin loss promotes experimental metastasis of squamous cell carcinoma

T-cadherin is gaining recognition as a determinant for the development of incipient invasive squamous cell carcinoma (SCC). However, effects of T-cadherin expression on the metastatic potential of SCC have not been studied. Here, using a murine model of experimental metastasis following tail vein injection of A431 SCC cells we report that loss of T-cadherin increased both the incidence and rate of appearance of lung metastases. T-cadherin-silenced SCC metastases were highly disordered with evidence of single cell dissemination away from main foci whereas SCC metastases overexpressing T-cadherin developed as compact, tightly organised sheets. SCC cell adhesion to vascular endothelial cells (EC) in culture was increased for T-cadherin-silenced SCC and decreased for T-cadherin-overexpressing SCC. Confocal microscopy showed that T-cadherin-silenced SCC adherent on EC display an elongated morphology with long thin extensions and a high degree of intercalation within the EC monolayer, whereas SCC overexpressing T-cadherin formed poorly-spread multicellular aggregates that remain on the outer surface of the EC monolayer. T-cadherin-deficient SCC or human keratinocyte cells exhibited increased transendothelial migration in vitro which could be attenuated in the presence of EGFR inhibitor gefitinib. Our data suggest that loss of T-cadherin can increase metastatic potential and aggressiveness of SCC, possibly due to facilitating arrest and extravasation through the vascular wall and/or more efficient establishment of metastases in the new microenvironment.

Angiographic geometric changes of the lumen arterial wall after bioresorbable vascular scaffolds and metallic platform stents at 1-year follow-up

The aim of this study was to compare the angiographic changes in coronary geometry of the bioresorbable vascular scaffolds (BVS) and metallic platform stent (MPS) between baseline and follow-up.

Dynamics of vessel wall changes following the implantation of the Absorb everolimus-eluting bioresorbable vascular scaffold: a multi-imaging modality study at 6, 12, 24 and 36 months

Aims: To assess observations with multimodality imaging of the Absorb bioresorbable everolimus-eluting vascular scaffold performed in two consecutive cohorts of patients who were serially investigated either at 6 and 24 months or at 12 and 36 months. Methods and results: In the ABSORB multicentre single-arm trial, 45 patients (cohort B1) and 56 patients (cohort B2) underwent serial invasive imaging, specifically quantitative coronary angiography (QCA), intravascular ultrasound (IVUS), radiofrequency backscattering (IVUS-VH) and optical coherence tomography (OCT). Between one and three years, late luminal loss remained unchanged (6 months: 0.19 mm, 1 year: 0.27 mm, 2 years: 0.27 mm, 3 years: 0.29 mm) and the in-segment angiographic restenosis rate for the entire cohort B (n=101) at three years was 6%. On IVUS, mean lumen, scaffold, plaque and vessel area showed enlargement up to two years. Mean lumen and scaffold area remained stable between two and three years whereas significant reduction in plaque behind the struts occurred with a trend toward adaptive restrictive remodelling of EEM. Hyperechogenicity of the vessel wall, a surrogate of the bioresorption process, decreased from 23.1% to 10.4% with a reduction of radiofrequency backscattering for dense calcium and necrotic core. At three years, the count of strut cores detected on OCT increased significantly, probably reflecting the dismantling of the scaffold; 98% of struts were covered. In the entire cohort B (n=101), the three-year major adverse cardiac event rate was 10.0% without any scaffold thrombosis. Conclusions: The current investigation demonstrated the dynamics of vessel wall changes after implantation of a bioresorbable scaffold, resulting at three years in stable luminal dimensions, a low restenosis rate and a low clinical major adverse cardiac events rate.

Thermal model for optimization of vascular laser tissue soldering

Laser tissue soldering (LTS) is a promising technique for tissue fusion based on a heat-denaturation process of proteins. Thermal damage of the fused tissue during the laser procedure has always been an important and challenging problem. Particularly in LTS of arterial blood vessels strong heating of the endothelium should be avoided to minimize the risk of thrombosis. A precise knowledge of the temperature distribution within the vessel wall during laser irradiation is inevitable. The authors developed a finite element model (FEM) to simulate the temperature distribution within blood vessels during LTS. Temperature measurements were used to verify and calibrate the model. Different parameters such as laser power, solder absorption coefficient, thickness of the solder layer, cooling of the vessel and continuous vs. pulsed energy deposition were tested to elucidate their impact on the temperature distribution within the soldering joint in order to reduce the amount of further animal experiments. A pulsed irradiation with high laser power and high absorbing solder yields the best results.

[Historical review and future orientations of the conventional vascular microanastomoses]

Microvascular surgery has become an important method for reconstructing surgical defects due to trauma, tumors or after burn. The most important factor for successful free flap transfer is a well-executed anastomosis. The time needed to perform the anastomosis and the failure rate are not negligible despite the high level of operator's experience. During the history, many alternatives were tried to help the microsurgeon and to reduce the complications. A Medline literature search was performed to find articles dealing with non-suture methods of microvascular anastomosis. Many historical books were also included. The non-suture techniques can be divided into four groups based on the used mechanism of sutures: double intubation including tubes and stents, intubation-eversion including simple rings, double eversion including staples and double rings, and wall adjustement with adhesives or laser. All these techniques were able to produce a faster and easier microvascular anastomosis. Nevertheless, disadvantages of the suturless techniques include toxicity, high cost, leakage or aneurysm formation. More refinement is needed before their widespread adoption. Thus, laser-assisted microvascular anastomosis using 1,9 μm diode laser appeared to be a safe and reliable help for the microsurgeon and may be further developed in the near future.

Differential distribution of vasa vasorum in different vascular beds in humans

OBJECTIVE: Vasa vasorum (VV) have been implicated to play a role in the pathogenesis of atherosclerosis. This study was designed to describe the distribution of VV density in different vascular beds in humans and to investigate the association between VV density and the known distribution of atherosclerosis in human arteries. METHODS: Forty-two human arteries, harvested at autopsy or after explantation, were analyzed by three-dimensional microscopic-computed tomography (micro-CT). VV density, endothelial-surface-fraction (Sigma VV endothelial-surface-area/vessel-wall-volume) and vascular-area-fraction (Sigma VV area/vessel-wall-area) were calculated for coronary, renal and femoral arteries. Representatively five coronary, renal and femoral arteries were stained for endothelial cells (von Willebrand-Factor), macrophages (CD68), vascular endothelial growth factor (VEGF) and collagen (Sirius Red). RESULTS: Coronary arteries showed a higher VV density compared to renal and femoral arteries (2.12+/-0.26 n/mm(2) versus 0.61+/-0.06 n/mm(2) and 0.66+/-0.11 n/mm(2); P<0.05 for both) as well as a higher endothelial-surface-fraction and vascular-area-fraction. Histology showed a positive correlation between histologically derived VV density and CD68-positive cells/area (r=0.54, P<0.01), VEGF-immunoreactivity/area (r=0.55, P<0.01) and a negative correlation between VV density and collagen I content (r=0.66, P<0.05). CONCLUSION: This micro-CT study highlights a higher VV density in coronary than in peripheral arteries, supporting the relation between VV density and the susceptibility to atherosclerosis in different vascular beds in humans.

Effects of an aging vascular model on healthy and diseased hearts

The vitamin D(3) and nicotine (VDN) model is a model of isolated systolic hypertension (ISH) due to arterial calcification raising arterial stiffness and vascular impedance similar to an aged and stiffened arterial tree. We therefore analyzed the impact of this aging model on normal and diseased hearts with myocardial infarction (MI). Wistar rats were treated with VDN (n = 9), subjected to MI by coronary ligation (n = 10), or subjected to a combination of both MI and VDN treatment (VDN/MI, n = 14). A sham-treated group served as control (Ctrl, n = 10). Transthoracic echocardiography was performed every 2 wk, whereas invasive indexes were obtained at week 8 before death. Calcium, collagen, and protein contents were measured in the heart and the aorta. Systolic blood pressure, pulse pressure, thoracic aortic calcium, and end-systolic elastance as an index of myocardial contractility were highest in the aging model group compared with MI and Ctrl groups (P(VDN) < 0.05, 2-way ANOVA). Left ventricular wall stress and brain natriuretic peptide (P(VDNxMI) = not significant) were highest, while ejection fraction, stroke volume, and cardiac output were lowest in the combined group versus all other groups (P(VDNxMI) < 0.05). The combination of ISH due to this aging model and MI demonstrates significant alterations in cardiac function. This model mimics several clinical phenomena of cardiovascular aging and may thus serve to further study novel therapies.

Sodium thiosulfate prevents vascular calcifications in uremic rats

Accelerated vascular calcification is a severe complication of chronic kidney disease contributing to high morbidity and mortality in patients undergoing renal replacement therapy. Sodium thiosulfate is increasingly used for the treatment of soft tissue calcifications in calciphylaxis. Therefore, we determined whether it also prevents development of vascular calcifications in chronic kidney disease. We found that uremic rats treated by thiosulfate had no histological evidence of calcification in the aortic wall whereas almost three-fourths of untreated uremic rats showed aortic calcification. Urinary calcium excretion was elevated and the calcium content of aortic, heart, and renal tissue was significantly reduced in the thiosulfate-treated compared to non-treated animals. Sodium thiosulfate treatment transiently lowered plasma ionized calcium and induced metabolic acidosis. It also lowered bone strength in the treated animals compared to their normal controls. Hence, sodium thiosulfate prevented vascular calcifications in uremic rats, likely by enhancing acid- and/or chelation-induced urinary calcium loss. The negative impact on rat bone integrity necessitates a careful risk-benefit analysis before sodium thiosulfate can be used in individual human patients.

High expression of gastrin-releasing peptide receptors in the vascular bed of urinary tract cancers: promising candidates for vascular targeting applications

Tumoral gastrin-releasing peptide (GRP) receptors are potential targets for diagnosis and therapy using radiolabeled or cytotoxic GRP analogs. GRP-receptor overexpression has been detected in endocrine-related cancer cells and, more recently, also in the vascular bed of selected tumors. More information on vascular GRP-receptors in cancer is required to asses their potential for vascular targeting applications. Therefore, frequent human cancers (n = 368) were analyzed using in vitro GRP-receptor autoradiography on tissue sections with the (125)I-[Tyr(4)]-bombesin radioligand and/or the universal radioligand (125)I-[d-Tyr(6), beta-Ala(11), Phe(13), Nle(14)]-bombesin(6-14). GRP-receptor expressing vessels were evaluated in each tumor group for prevalence, quantity (vascular score), and GRP-receptor density. Prevalence of vascular GRP-receptors was variable, ranging from 12% (prostate cancer) to 92% (urinary tract cancer). Different tumor types within a given site had divergent prevalence of vascular GRP-receptors (e.g. lung: small cell cancer: 0%; adenocarcinoma: 59%; squamous carcinoma: 83%). Also the vascular score varied widely, with the highest score in urinary tract cancer (1.69), moderate scores in lung (0.91), colon (0.88), kidney (0.84), and biliary tract (0.69) cancers and low scores in breast (0.39) and prostate (0.14) cancers. Vascular GRP-receptors were expressed in the muscular vessel wall in moderate to high densities. Normal non-neoplastic control tissues from these organs lacked vascular GRP-receptors. In conclusion, tumoral vessels in all evaluated sites express GRP-receptors, suggesting a major biological function of GRP-receptors in neovasculature. Vascular GRP-receptor expression varies between the tumor types indicating tumor-specific mechanisms in their regulation. Urinary tract cancers express vascular GRP-receptors so abundantly, that they are promising candidates for vascular targeting applications.