Isolation of Primary Mouse Pulmonary Microvascular Endothelial Cells and Generation of an Immortalized Cell Line to Obtain Sufficient Extracellular Vesicles.

Pulmonary microvascular endothelial cells (PMECs) and the extracellular vesicles (EVs) derived from PMECs participate in maintaining pulmonary homeostasis and mediating the inflammatory response. However, obtaining a high-purity population of PMECs and their EVs from mouse is still notoriously difficult. Herein we provide a method to isolate primary mouse PMECs (pMPMECs) and to transduce SV40 lentivirus into pMPMECs to establish an immortalized cell line (iMPMECs), which provides sufficient quantities of EVs for further studies. pMPMECs and iMPMECs can be identified using morphologic criteria, a phenotypic expression profile (e.g., CD31, CD144, G. simplicifolia lectin binding), and functional properties (e.g., Dil-acetylated low-density protein uptake, Matrigel angiogenesis). Furthermore, pMPMEC-EVs and iMPMEC-EVs can be identified and compared. The characteristics of pMPMEC-EVs and iMPMEC-EVs are ascertained by transmission electron microscopy, nanoparticle tracking analysis, and specific protein markers. iMPMECs produce far more EVs than pMPMECs, while their particle size distribution is similar. Our detailed protocol to isolate and immortalize MPMECs will provide researchers with an in vitro model to investigate the specific roles of EVs in pulmonary physiology and diseases.

Bioengineered 3D Microvessels for Investigating Plasmodium falciparum Pathogenesis.

Plasmodium falciparum pathogenesis is complex and intimately connected to vascular physiology. This is exemplified by cerebral malaria (CM), a neurovascular complication that accounts for most of the malaria deaths worldwide. P. falciparum sequestration in the brain microvasculature is a hallmark of CM and is not replicated in animal models. Numerous aspects of the disease are challenging to fully understand from clinical studies, such as parasite binding tropism or causal pathways in blood-brain barrier breakdown. Recent bioengineering approaches allow for the generation of 3D microvessels and organ-specific vasculature that provide precise control of vessel architecture and flow dynamics, and hold great promise for malaria research. Here, we discuss recent and future applications of bioengineered microvessels in malaria pathogenesis research.

Long non­coding RNA SNHG16 inhibits the oxygen­glucose deprivation and reoxygenation­induced apoptosis in human brain microvascular endothelial cells by regulating miR­15a­5p/bcl­2.

MicroRNA (miR) 15a­5p can promote ischemia/reperfusion (I/R)­induced apoptosis of cerebral vascular endothelial cells, which is inhibited by long non­coding RNAs (lncRNAs). The present study investigated the potential of lncRNAs targeting miR­15a­5p to regulate oxygen­glucose deprivation and reoxygenation (OGD­R)­induced apoptosis of human brain microvascular endothelial cells (hBMECs). hBMECs were transfected with or without miR­15a­5p or its mutant, together with p­small nucleolar RNA host gene 16 (SNHG16) or its mutant. Following OGD­R, proliferation, apoptosis and miR­15a­5p, SNHG16 and Bcl­2 expression levels were determined using MTT, flow cytometry, reverse transcription­quantitative PCR or western blotting. The potential interaction of SNHG16 with miR­15a­5p was analyzed by pull­down, luciferase and immunoprecipitation assays. OGD­R induced apoptosis of hBMECs and increased miR­15a­5p expression levels in a time­dependent manner. miR­15a­5p overexpression decreased the proliferation of hBMECs and promoted apoptosis by decreasing Bcl­2 expression levels. SNHG16 was pulled­down by miR­15a­5p and anti­Ago2. miR­15a­5p overexpression significantly decreased SNHG16­regulated luciferase activity and hBMEC survival by increasing apoptosis. SNHG16 overexpression decreased miR­15a­5p expression levels in hBMECs. SNHG16 gradually decreased following OGD­R and its overexpression decreased miR­15a­5p expression levels and promoted the proliferation of hBMECs by decreasing apoptosis. SNHG16 enhanced Bcl­2 expression levels in hBMECs, which was abrogated by miR­15a­5p. Bioinformatics suggest that SNHG16 may antagonize the binding of miR­15a­5p to the 3'UTR of Bcl­2 mRNA. These findings suggest that SNHG16 may protect hBMECs from OGD­R­induced apoptosis by antagonizing the miR­15a­5p/bcl­2 axis. Thus, targeting SNHG16­based mechanisms may provide novel therapeutic strategies for treatment of ischemic stroke.

The isolation and molecular characterization of cerebral microvessels.

The study of cerebral microvessels is becoming increasingly important in a wide variety of conditions, such as stroke, sepsis, traumatic brain injury and neurodegenerative diseases. However, the molecular mechanisms underlying cerebral microvascular dysfunction in these conditions are largely unknown. The molecular characterization of cerebral microvessels in experimental disease models has been hindered by the lack of a standardized method to reproducibly isolate intact cerebral microvessels with consistent cellular compositions and without the use of enzymatic digestion, which causes undesirable molecular and metabolic changes. Herein, we describe an optimized protocol for microvessel isolation from mouse brain cortex that yields microvessel fragments with consistent populations of discrete blood-brain barrier (BBB) components (endothelial cells, pericytes and astrocyte end feet) while retaining high RNA integrity and protein post-translational modifications (e.g., phosphorylation). We demonstrate that this protocol allows the quantification of changes in gene expression in a disease model (stroke) and the activation of signaling pathways in mice subjected to drug administration in vivo. We also describe the isolation of genomic DNA (gDNA) and bisulfite treatment for the assessment of DNA methylation, as well as the optimization of chromatin extraction and shearing from cortical microvessels. This optimized protocol and the described applications should improve the understanding of the molecular mechanisms governing cerebral microvascular dysfunction, which may help in the development of novel therapies for stroke and other neurologic conditions.

Atmospheric nanoparticles affect vascular function using a 3D human vascularized organotypic chip.

Inhaled atmospheric nanoparticles (ANPs) can migrate into human blood vessels. However, the exact pathogenesis has not yet been well elucidated. In this study, a perfusable 3D human microvessel network was constructed in a microfluidic device. This functional 3D micro-tissue partly mimicked the physiological response of human vessels. Intravascular nanoparticles tend to adsorb proteins to form a protein corona. Based on this pathological response, vessel permeability and vasoconstriction resulting from ANP stimulation might be related to vascular inflammation. It mediated abnormal expression of nuclear factor-κB (NF-κB) and an influx of intracellular Ca2+ ([Ca2+]i). This biological behavior disturbed the normal expression of intercellular cell adhesion molecule 1 (ICAM-1) and vascular endothelial growth factor (VEGF). The imbalance of nitric oxide (NO) and endothelin-1 (ET-1) further resulted in endothelial cell contraction. All these bio-events induced the loss of tight junctions (ZO-1) which enhanced vessel permeability. Meanwhile, ANP induced-vascular toxicity was also found in mice. Our observations provide a plausible explanation for how the ANPs affect human vascular function. The vessel-on-chip provides a bridge between in vitro results and human responses. We aimed to use this human 3D functional microvascular model to mimic the physiological responses of human vessels. This model is suitable for the evaluation of vascular toxicity after the human vessel exposure to ANPs.

The neuritic plaque in Alzheimer's disease: perivascular degeneration of neuronal processes.

Cerebrovascular pathology is common in aging and Alzheimer's disease (AD). The microvasculature is particularly vulnerable, with capillary-level microhemorrhages coinciding with amyloid beta deposits in senile plaques. In the current analysis, we assessed the relationship between cerebral microvessels and the neuritic component of the plaque in cortical and hippocampal 50- to 200-µm sections from 11 AD, 3 Down syndrome, and 7 nondemented cases in neuritic disease stages 0-VI. We report that 77%-97% of neuritic plaques are perivascular, independently of disease stage or dementia diagnosis. Within neuritic plaques, dystrophic hyperphosphorylated tau-positive neurites appear as clusters of punctate, bulbous, and thread-like structures focused around capillaries and colocalize with iron deposits characteristic of microhemorrhage. Microvessels within the neuritic plaque are narrowed by 1.0 ± 1.0 µm-4.4 ± 2.0 µm, a difference of 16%-65% compared to blood vessel segments with diameters 7.9 ± 2.0-6.4 ± 0.8 µm (p < 0.01) outside the plaque domain. The reduced capacity of microvessels within plaques, frequently below patency, likely compromises normal microlocal cerebrovascular perfusion. These data link the neuritic and amyloid beta components of the plaque directly to microvascular degeneration. Strategies focused on cerebrovascular antecedents to neuritic dystrophy in AD have immediate potential for prevention, detection, and therapeutic intervention.

Anatomy, immunohistochemistry, and numerical distribution of human splenic microvessels.

The microvascular architecture of the spleen plays an important role in the immunological function of this organ. The different types of vessels are related to different reticular cells each with their own immunomodulatory functions. The present study describes an immunohistochemical and morphometric analysis of the various types of vessels in 21 human autopsy non-pathological splenic samples. On an area of 785,656.37 µm2 for each sample, we classified and quantified the type and number of vascular structures, each according to their morphology and immunohistochemical profile, and obtained the ratios between them. The distribution of trabecular vessels and the characteristics of the venules are reviewed. In our material the so-called "cavernous perimarginal sinus" (anatomical structure previously described by Schmidt et al., 1988) was observed and interpreted as a curvilinear venule shaped by the follicle in contact with the trabecular vein. Our material comprised 261 trabeculae (containing 269 arterial sections and 508 venous sections), 30,621 CD34+ capillaries, 7739 CD271+ sheathed capillaries, 2588 CD169+ sheathed capillaries, and 31,124 CD8+ sinusoids. The total area (TA) (14,765,714.88 µm2) occupied by the sinusoidal sections of the 21 cases was much higher than the TA of the capillary sections (1,700,269.83 µm2). Similarly, the TA (651,985 µm2) occupied by the sections of the trabecular veins was much higher than the TA of the trabecular arteries (88,594 µm2). The total number of CD34+ capillaries and of sinusoids CD8+ was similar for the sum of the 21 cases, nevertheless there were large differences in each case. Statistically the hypothesis that the number of capillaries and sinusoids are present with the same frequency is discarded. In view of the absence of a numerical correlation between capillaries and sinusoids, we postulate that very possibly the arterial and the venous vascular trees are two anatomically independent structures separated by the splenic cords. We believe that this is the first work where splenic microvascularization is simultaneously approached from a morphometric and immunohistochemical point of view.

Pro-angiogenic Ginsenosides F1 and Rh1 Inhibit Vascular Leakage by Modulating NR4A1.

Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, but VEGF-induced angiogenesis is often accompanied by a vascular permeability response. Ginsenosides are triterpenoid saponins from the well-known medicinal plant, ginseng, and have been considered a candidate for modulating angiogenesis. Here, we systemically investigated the effects of 10 different ginsenosides on human umbilical vein endothelial cells and newly identified that two PPT-type ginsenosides, F1 and Rh1 induce the migration and proliferation of endothelial cells. Interestingly, RNA transcriptome analysis showed that gene regulation induced by VEGF in endothelial cells is distinct from that of ginsenoside F1 and Rh1. In addition, F1 and Rh1 significantly inhibited vascular leakage both in vitro and in vivo, which are induced by vascular endothelial growth factor. Furthermore, comparative transcriptome analysis revealed that these effects of F1 and Rh1 on vascular leakage restoration are mainly caused by changes in VEGF-mediated TNFα signaling via NFκB, particularly by the suppression of expression and transcriptional activity of NR4A1 by F1 and Rh1, even in the presence of VEGF. These findings demonstrate that ginsenosides F1 and Rh1 can be a promising herbal remedy for vessel normalization in ischemic disease and cancer and that NR4A1 is the key target.

Microvessel density in breast cancer: the impact of field area on prognostic informativeness.

AIMS: Tumour microvessel density (MVD) is assessed by counting vessels in the most vascularised tumour region, the vascular hot spot. Current uncertainty regarding the prognostic role of MVD in breast cancer could, in part, be explained by variations in field area size for MVD assessment. We aimed to identify the field area size that provides the most accurate prognostic information in breast carcinoma. METHODS: MVD was assessed in 212 tumours. von Willebrand factor positively stained vessels were counted in 10 consecutive visual fields in vascular hotspots. The 10 visual fields in the original counting sequence (MVD-Consecutive) were sorted from highest to lowest vessel count (MVD-Decreasing), and randomly (MVD-Random). After adding counts from one visual field at a time, mean MVD was calculated for each cumulative field area. The prognostic informativeness of each field area and sorting strategy were compared. RESULTS: Median MVD decreased with increasing field size for MVD-Decreasing and MVD-Consecutive. A 0.35 mm2 total field area comprising only the highest vessel counts provided the most accurate prognostic information (MVD-Decreasing, HR for breast cancer death 1.06 per 10 vessels/mm2 increase, 95% CI 1.03 to 1.10). MVD-Decreasing gave more accurate prognostic information than MVD-Consecutive and MVD-Random, with decreasing prognostic informativeness with increasing field area. CONCLUSIONS: Median MVD and its prognostic informativeness decreased with increasing field area. Assessing MVD in a carefully selected small field area of 0.35 mm2 provides the most accurate prognostic information. This could facilitate the implementation of MVD assessment in breast cancer.

Threshold-dependent nonlinear scattering from porphyrin nanobubbles for vascular and extravascular applications.

Nanobubbles hold potential for expanding utility of ultrasound contrast-based applications to extravascular targets, but their acoustic response and the effects of the surrounding environment remain relatively unexplored. Here we investigate the dynamics of porphyrin-encapsulated nanobubbles (diameter <0.4 µm; 106 ml-1) at clinically relevant frequencies (2.5 MHz and 8 MHz) as a function of pressure (0.1-1.0 MPa) in vessel- and tissue-mimicking phantoms to gain an understanding of nanobubble behaviour in intra- and extravascular compartments. The results provide the first direct observation that nanobubbles can initiate nonlinear scattering, and that they do so in a pressure-dependent manner. It is further demonstrated that while nanobubbles in confining media require higher pressures for nonlinearities and demonstrate reduced scattering, they can exhibit sustained and non-destructive cavitation. Bubble models are then used to gain mechanistic insights into experimentally observed nanobubble dynamics and confirm sensitivity to nonlinear shell rheology, particularly to radially-dependent surface tension and the characteristic time constant for shear-thinning.

Antibody Cross-Linking of CD14 Activates MerTK and Promotes Human Macrophage Clearance of Apoptotic Neutrophils: the Dual Role of CD14 at the Crossroads Between M1 and M2c Polarization.

Mer receptor tyrosine kinase (MerTK) is key for efficient phagocytosis of apoptotic neutrophils (ANs) and homeostasis of IL-10 production by human anti-inflammatory M2c monocytes/macrophages. We asked whether stimulation of M2c surface receptors contributes in turn to MerTK activation. For this purpose, human monocytes/macrophages were differentiated under M1, M2a, and M2c polarizing conditions. The effects of antibody-mediated cross-linking of M2c receptors (i.e., CD14, CD16, CD32, CD163, CD204) on MerTK phosphorylation and phagocytosis of ANs were tested. MerTK expression was also studied by flow cytometry and western blot in the presence of LPS and in M2c-derived microvesicles (MVs). Antibody cross-linking of either CD14 or CD32/FcγRII led to Syk activation and MerTK phosphorylation in its two distinct glycoforms (175-205 and 135-155 kDa). Cross-linked CD14 enhanced efferocytosis by M2c macrophages and enabled M1 and M2a cells to clear ANs efficiently. In M1 conditions, LPS abolished surface MerTK expression on CD14bright cell subsets, so disrupting the anti-inflammatory pathway. In M2c cells, instead, MerTK was diffusely and brightly co-expressed with CD14, and was also detected in M2c macrophage-derived MVs; in these conditions, LPS only partially downregulated MerTK on cell surfaces, while the smaller MerTK glycoform contained in MVs remained intact. Altogether, cooperation between CD14 and MerTK may foster the clearance of ANs by human monocytes/macrophages. CD14 stands between M1-related LPS co-receptor activity and M2c-related MerTK-dependent response. MerTK interaction with CD32/FcγRII, its detection in M2c MVs, and the differential localization and LPS susceptibility of MerTK glycoforms add further new elements to the complexity of the MerTK network.

Liver segment imaging using monocyte sequestration: a potential tool for fluorescence-guided liver surgery.

Background: The accurate determination of liver segment anatomy is essential to perform liver resection without complications and to ensure long-term outcomes after this operation. There are several perioperative methods for segment identification and surgical navigation, such as intraoperative ultrasound, indigo carmine and near-infrared imaging with indocyanine green. The present study experimentally analyzed the usefulness of monocyte sequestration for liver segment labeling and imaging. Methods: Human monocytes were isolated from peripheral blood and directly or indirectly labeled with calcein or IRDye 800CW. Potential toxicity, labeling stability, and adhesion to ICAM-1 were analyzed in vitro. Monocyte sequestration in the liver microvasculature and liver segment labeling and boundary demarcation were studied using isolated mouse and pig liver perfusion and via intraportal injection in mouse liver tumor models. Results: The highest monocyte labeling efficiency was achieved using direct labeling with IRDye 800CW. Labeling was stable and did not influence cell viability. The labeled monocytes were highly sequestrated in the liver microvasculature, both after ex vivo perfusion and after injection in vivo , resulting in excellent labeling of selected liver segments and strong segment boundary demarcation. In contrast to results to a normal liver, monocyte sequestration was very low in tumor-associated blood vessels. Conclusions: The present experimental study shows that sequestration of labeled monocytes after superselective application demarcates the selected liver segment. These results illustrate potential of this technique for surgical navigation during liver surgery.

Radiologically Undetected Hepatocellular Carcinoma in Patients Undergoing Liver Transplantation: An Immunohistochemical Correlation With LI-RADS Score.

Orthotopic liver transplantation is the best option for patients with carefully selected unresectable disease because of underlying liver dysfunction. The 5-year survival rate after orthotopic liver transplantation for early detected hepatocellular carcinoma (HCC) is high, and a similar or even higher rate is reported in those with radiologically undetected HCC. This study evaluated and compared the histologic features of pretransplant radiologically undetected (14 patients, 25 tumors) versus detected (36 patients, 45 tumors) HCCs. Tumor size, tumor differentiation, number of unpaired arteries, mitotic count per 10 high-power fields, CD34 immunostain to assess microvessel density, and Ki67 immunostain were compared with the Liver Imaging Reporting and Data System score, which was retrospectively assigned to each tumor in both groups. The Liver Imaging Reporting and Data System score was significantly higher in the HCC detected group (P<0.001). The vast majority of the undetected HCCs (88%) was <2 cm in size. Only 12% of the undetected HCCs were ≥2 cm, whereas 51% of the detected HCCs were ≥2 cm in size. Higher rate of moderate to poor tumor differentiation was noted in the detected HCCs compared with the undetected group (89% vs. 60%; P=0.004). No statistically significant difference in the number and distribution of unpaired arteries, or mitotic count was observed in 2 groups (although fewer unpaired arteries were identified in the undetected group). The detected HCCs had a higher rate of 2+ CD34 staining compared with the undetected HCCs (68% vs. 27%; P=0.002), whereas the opposite was observed for 1+ CD34 staining (59% undetected HCCs vs. 17% detected HCCs; P=0.002). Ki67 proliferative index was not statistically different between the 2 groups (120.8/1000 cells detected HCCs vs. 81.8/1000 cells undetected HCCs; P=0.36). The factors associated with failing to detect HCCs pretransplant by radiologic studies include small tumor size (<2 cm), low-grade histologic differentiation, and low microvessel density (low CD34 staining). A significant association between the number and distribution of unpaired arteries and HCC detection has not been established by our study.

Regulation of cerebral arterial BKCa channels by angiotensin II signaling in adult offspring exposed to prenatal high sucrose diets.

Prenatal insults have been shown to affect vascular functions, leading to increased risks of cardiovascular diseases in offspring. The present study determined whether high sucrose (HS) intake in pregnancy affected central vascular functions in middle cerebral artery (MCA) of offspring. Sprague-Dawley rats were fed a standard food and tap water with normal or high (20%) sucrose content during pregnancy. Offspring were maintained with normal diets and tap water. Central vascular functions and related ion channels were assessed in male offspring at 5 months old. Compared with the control, angiotensin II (AII)-induced vasoconstrictions were significantly higher in the MCA of the offspring exposed to prenatal HS. In the MCA, large conductance Ca2+-activated K+ channels (BKCa) currents were decreased with a reduction of opening frequency, sensitivity to intracellular Ca2+/membrane voltage, and BKß1 expression. mRNA levels of AT1α and AT2, as well as AT1/AT2 ratio, were significantly increased in the MCA of offspring following exposure to prenatal HS diets. The data suggested that prenatal HS diets could alter microvascular activities in the MCA, probably via changes of BKCa channels in the brain.

Nuclear survivin expression correlates with endoglin-assessed microvascularisation in laryngeal carcinoma.

AIMS: Survivin-a member of the family of inhibitor of apoptosis proteins that control cell division, apoptosis and metastasis-is overexpressed in virtually all human cancers, including laryngeal squamous cell carcinoma (LSCC). Recent findings also correlate survivin expression with the regulation of angiogenesis. The novel main aim of this study was a preliminary investigation into the potential role of survivin expression in LSCC neoangiogenesis, as determined by endoglin-assessed microvascular density (MVD). METHODS: Immunohistochemical expression of nuclear survivin and endoglin-assessed MVD were ascertained by image analysis in 75 consecutive LSCCs. RESULTS: Statistical analysis disclosed a strong direct correlation between nuclear survivin expression and MVD. Patients whose nuclear survivin expression was ≥6.0% had a significantly higher LSCC recurrence rate, and a significantly shorter disease-free survival (DFS) than those with a nuclear survivin expression <6.0%. The LSCC recurrence rate was also higher and the DFS shorter in patients with endoglin-assessed MVD ≥6.89%. The OR for recurrence was 2.79 in patients with LSCC with a nuclear survivin expression ≥6.0%, and 12.31 in those with an MVD≥6.89%. CONCLUSIONS: Survivin-targeting strategies to enhance tumour cell response to apoptosis and inhibit tumour growth should receive more attention with a view to developing agents for use in multimodality advanced LSCC treatment, or combined with conventional chemotherapy. Given the present preliminary evidence in LSCC, survivin targeting should also be further investigated for anti-angiogenic purposes, to reduce tumour blood flow and induce cancer necrosis.

Quantifying tumour vascularity in non-luminal breast cancers.

AIMS: Microvessel density (MVD), proliferating MVD (pMVD) and vascular proliferation index (VPI) are methods used to quantify tumour vascularity in histopathological sections. In this study, we assessed MVD, pMVD and VPI in non-luminal subtypes of breast cancer. Differences between subtypes were studied, and the prognostic value of each method was assessed. METHODS: All non-luminal subtypes (61 basal phenotype (BP), 60 human epidermal growth factor receptor 2 (HER2) type and 30 five negative phenotype (5NP)) were selected from a series comprising 909 cases of breast cancer. Sections were stained for Ki67 and von Willebrand factor. Associations between MVD, pMVD and VPI, molecular subtypes and prognosis were studied. RESULTS: MVD was highest in 5NP (Δ54.3 microvessels/mm2 compared with BP, 95% CI 30.3 to 78.3), whereas no clear difference was found between HER2 type and BP (Δ8.8 microvessels/mm2, 95% CI -9.6 to 27.1). pMVD and VPI did not differ between subtypes. For MVD, HR was 1.07 (95% CI 1.03 to 1.11) per 10 vessel increase and 1.9 (95% CI 1.2 to 3.1) if MVD was greater than median value. High MVD was associated with poor prognosis in the HER2 type (HR 1.07 (95% CI 1.02 to 1.12)) and 5NP (HR 1.13 (95% CI 1.03 to 1.23)), but not in BP (HR 1.04 (95% CI 0.94 to 1.14) per 10 vessel increase). pMVD and VPI were not associated with prognosis. CONCLUSIONS: MVD appears to be an independent prognostic factor in HER2 and 5NP subtypes of breast cancer, where high MVD is associated with poor survival. MVD was higher in the 5NP compared with both BP and HER2 type.

Endothelial-to-mesenchymal transition contributes to endothelial dysfunction and dermal fibrosis in systemic sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) features multiorgan fibrosis orchestrated predominantly by activated myofibroblasts. Endothelial-to-mesenchymal transition (EndoMT) is a transdifferentiation by which endothelial cells (ECs) lose their specific morphology/markers and acquire myofibroblast-like features. Here, we determined the possible contribution of EndoMT to the pathogenesis of dermal fibrosis in SSc and two mouse models. METHODS: Skin sections were immunostained for endothelial CD31 or vascular endothelial (VE)-cadherin in combination with α-smooth muscle actin (α-SMA) myofibroblast marker. Dermal microvascular ECs (dMVECs) were prepared from SSc and healthy skin (SSc-dMVECs and H-dMVECs). H-dMVECs were treated with transforming growth factor-ß1 (TGFß1) or SSc and healthy sera. Endothelial/mesenchymal markers were assessed by real-time PCR, immunoblotting and immunofluorescence. Cell contractile phenotype was assayed by collagen gel contraction. RESULTS: Cells in intermediate stages of EndoMT were identified in dermal vessels of either patients with SSc or bleomycin-induced and urokinase-type plasminogen activator receptor (uPAR)-deficient mouse models. At variance with H-dMVECs, SSc-dMVECs exhibited a spindle-shaped appearance, co-expression of lower levels of CD31 and VE-cadherin with myofibroblast markers (α-SMA+ stress fibres, S100A4 and type I collagen), constitutive nuclear localisation of the EndoMT driver Snail1 and an ability to effectively contract collagen gels. Treatment of H-dMVECs either with SSc sera or TGFß1 resulted in the acquisition of a myofibroblast-like morphology and contractile phenotype and downregulation of endothelial markers in parallel with the induction of mesenchymal markers. Matrix metalloproteinase-12-dependent uPAR cleavage was implicated in the induction of EndoMT by SSc sera. CONCLUSIONS: In SSc, EndoMT may be a crucial event linking endothelial dysfunction and development of dermal fibrosis.

The role of the microvascular network structure on diffusion and consumption of anticancer drugs.

We investigate the impact of microvascular geometry on the transport of drugs in solid tumors, focusing on the diffusion and consumption phenomena. We embrace recent advances in the asymptotic homogenization literature starting from a double Darcy-double advection-diffusion-reaction system of partial differential equations that is obtained exploiting the sharp length separation between the intercapillary distance and the average tumor size. The geometric information on the microvascular network is encoded into effective hydraulic conductivities and diffusivities, which are numerically computed by solving periodic cell problems on appropriate microscale representative cells. The coefficients are then injected into the macroscale equations, and these are solved for an isolated, vascularized spherical tumor. We consider the effect of vascular tortuosity on the transport of anticancer molecules, focusing on Vinblastine and Doxorubicin dynamics, which are considered as a tracer and as a highly interacting molecule, respectively. The computational model is able to quantify the treatment performance through the analysis of the interstitial drug concentration and the quantity of drug metabolized in the tumor. Our results show that both drug advection and diffusion are dramatically impaired by increasing geometrical complexity of the microvasculature, leading to nonoptimal absorption and delivery of therapeutic agents. However, this effect apparently has a minor role whenever the dynamics are mostly driven by metabolic reactions in the tumor interstitium, eg, for highly interacting molecules. In the latter case, anticancer therapies that aim at regularizing the microvasculature might not play a major role, and different strategies are to be developed.

Image-enhanced bronchoscopic evaluation of bronchial mucosal microvasculature in COPD.

BACKGROUND: Bronchial vascular remodeling is an underresearched component of airway remodeling in COPD. Image-enhanced bronchoscopy may offer a less invasive method for studying bronchial microvasculature in COPD. OBJECTIVES: To evaluate endobronchial mucosal vasculature and changes in COPD by image-enhanced i-scan3 bronchoscopy and correlate them pathologically by analyzing bronchial mucosal biopsies. METHODS: This case-control study analyzed 29 COPD patients (41.4% Global initiative for chronic Obstructive Lung Disease B [GOLD B] and 58.6% GOLD D) and ten healthy controls admitted at Alexandria Main University Hospital, Egypt. Combined high-definition white light bronchoscopy (HD WLB) with i-scan3 was used to evaluate endobronchial mucosal microvasculature. The vascularity was graded according to the level of mucosal red discoloration (ie, endobronchial erythema) from decreased discoloration to normal, mild, moderate, and severe increased red discoloration (G-1, G0, G+1, G+2, and G+3, respectively) and scored by three bronchoscopists independently. Bronchial mucosal biopsies were taken for microvascular density counting using anti-CD34 antibody as angiogenesis marker. RESULTS: Different grades of endobronchial erythema were observed across/within COPD patients using combined HD WLB + i-scan3, with significant agreement among scorers (P=0.031; median score of G+1 [G-1-G+2]) being higher in GOLD D (P=0.001). Endobronchial erythema significantly correlated with COPD duration, exacerbation frequency, and body mass index (P<0.05). Angiogenesis was significantly decreased among COPD patients versus controls (10.6 [8-13.3] vs 14 [11-17.1]; P=0.02). Mucosal surface changes (including edema, atrophy, and nodules) were better visualized by the combined HD WLB + i-scan3 rather than HD WLB alone. CONCLUSION: Combined HD WLB + i-scan3 seems to be valuable in evaluating mucosal microvasculature and surface changes in COPD, which may represent vasodilatation rather than angiogenesis.

Investigating the effect of tumor vascularization on magnetic targeting in vivo using retrospective design of experiment.

Nanocarriers take advantages of the enhanced permeability and retention (EPR) to accumulate passively in solid tumors. Magnetic targeting has shown to further enhance tumor accumulation in response to a magnetic field gradient. It is widely known that passive accumulation of nanocarriers varies hugely in tumor tissues of different tumor vascularization. It is hypothesized that magnetic targeting is likely to be influenced by such factors. In this work, magnetic targeting is assessed in a range of subcutaneously implanted murine tumors, namely, colon (CT26), breast (4T1), lung (Lewis lung carcinoma) cancer and melanoma (B16F10). Passively- and magnetically-driven tumor accumulation of the radiolabeled polymeric magnetic nanocapsules are assessed with gamma counting. The influence of tumor vasculature, namely, the tumor microvessel density, permeability and diameter on passive and magnetic tumor targeting is assessed with the aid of the retrospective design of experiment (DoE) approach. It is clear that the three tumor vascular parameters contribute greatly to both passive and magnetically targeted tumor accumulation but play different roles when nanocarriers are targeted to the tumor with different strategies. It is concluded that tumor permeability is a rate-limiting factor in both targeting modes. Diameter and microvessel density influence passive and magnetic tumor targeting, respectively.