Postcapillary Venules Research Articles

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay.

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

Transcriptomic Analysis Reveals Differential Expression of Genes between Lung Capillary and Post Capillary Venules in Abdominal Sepsis.

Lung endothelial cell dysfunction plays a central role in septic-induced lung injury. We hypothesized that endothelial cell subsets, capillary endothelial cells (capEC) and post capillary venules (PCV), might play different roles in regulating important pathophysiology in sepsis. In order to reveal global transcriptomic changes in endothelial cell subsets during sepsis, we induced sepsis in C57BL/6 mice by cecal ligation and puncture (CLP). We confirmed that CLP induced systemic and lung inflammation in our model. Endothelial cells (ECs) from lung capillary and PCV were isolated by cell sorting and transcriptomic changes were analyzed by bioinformatic tools. Our analysis revealed that lung capEC are transcriptionally different than PCV. Comparison of top differentially expressed genes (DEGs) of capEC and PCV revealed that capEC responses are different than PCV during sepsis. It was found that capEC are more enriched with genes related to regulation of coagulation, vascular permeability, wound healing and lipid metabolic processes after sepsis. In contrast, PCV are more enriched with genes related to chemotaxis, cell–cell adhesion by integrins, chemokine biosynthesis, regulation of actin filament process and neutrophil homeostasis after sepsis. In addition, we predicted some transcription factor targets that regulate a significant number of DEGs in sepsis. We proposed that targeting certain DEGs or transcriptional factors would be useful in protecting against sepsis-induced lung damage.

Protective effects of FK 506 against haemorrhagic shock-induced microvascular hyperpermeability.

Microvascular hyperpermeability, the excessive leakage of fluid and proteins from the intravascular space to the interstitium, is a devastating clinical concern in haemorrhagic shock (HS), sepsis, burn and so forth. Previous studies have shown that HS-induced microvascular hyperpermeability is associated with activation of the mitochondria-mediated 'intrinsic' apoptotic signalling cascade and caspase-3 mediated disruption of the endothelial cell barrier. In this study, our objective was to test if FK506, an immunomodulator that is also known to protect mitochondria, would protect barrier functions and decrease vascular hyperpermeability following HS by acting on this pathway. FK506 (25µM) was given 10 minutes before the shock period in a rat model of HS. The HS model was a non-traumatic/fixed pressure model of hypovolemic shock developed by withdrawing blood to reduce the mean arterial pressure to 40mm Hg for 60minutes. The mesenteric post-capillary venules were monitored for changes in permeability using intravital microscopic imaging. The changes in mitochondrial transmembrane potential (MTP) were determined using the cationic dye 5,5',6,6' tetrachoro-1,1',3,3' tetraethyl benzimidazolyl carbocyanine iodide (JC-1), that was superfused on the mesenteric vasculature followed by intravital imaging. The mesenteric caspase-3 activity was measured fluorometrically. Haemorrhagic shock induced a significant increase in hyperpermeability compared to the sham-control group and FK506 treatment decreased HS-induced hyperpermeability significantly (P<.05). FK506 dampened HS-induced loss of MTP and elevation of caspase-3 activity significantly (P<.05). FK506 has protective effects against HS-induced microvascular hyperpermeability. The maintenance of the MTP and protection against caspase-3 mediated endothelial cell barrier disruption are possible mechanisms by which FK506 attenuates HS-induced hyperpermeability. FK506, currently used in clinical settings as an immunomodulator, needs to be explored further for its therapeutic usefulness against HS-induced vascular hyperpermeability and associated complications.

Abstract P509: Novel Protein Kinase C Epsilon Inhibitor Attenuates Uncoupled Endothelial Nitric Oxide Synthase And Vascular Endothelial Dysfunction

Endothelial nitric oxide synthase (eNOS) uncoupling reduces nitric oxide (NO) production when tetrahydrobiopterin (BH 4 ) is oxidized to dihydrobiopterin (BH 2 ), resulting in endothelial dysfunction and leukocyte adherence. Renovascular damage following shockwave lithotripsy (SWL) may occur via the potentiation of this mechanism. We aim to modulate uncoupled eNOS activity with myristoylated protein kinase C epsilon inhibitor ( myr -PKCε–; N- myr -EAVSLKPT) and activator ( myr -PKCε+; N- myr -HDAPIGYD). We hypothesize that myr -PKCε– with uncoupled eNOS (i.e., bound to BH 2 ) should reduce leukocyte-endothelial interactions in postcapillary venules and H 2 O 2 levels after SWL. Myr -PKCε+ should promote similar effects when eNOS is coupled (i.e., bound to BH 4 ). Mesenteric venules of anesthetized male SD rats (300g) were superfused for 120 min with 100 μM BH 2 and test solutions (Krebs’ buffer, 10 μM myr -PKCε+, or 10 μM myr -PKCε–) with or without 100 μM BH 4 . Leukocyte activity was evaluated via intravital microscopy. In separate SD rats, SWL was performed on left kidneys (16 kV, 500 shocks at 60 shocks/min and at 120 shocks/min). Then, normal saline or test solution cocktail ( myr -PKCε+ [0.9 mg/kg]/ myr -PKCε- [0.8 mg/kg] with BH 2 [2mg/kg] or BH 4 [0.8 mg/kg]) was given i.v. NO or H 2 O 2 levels were measured with 100 μm microsensors in left renal veins at baseline, end of SWL, and 5-min intervals for 30 min post-SWL. Data were analyzed using ANOVA Student-Newman-Keuls test. BH 2 -induced leukocyte rolling, adherence, and transmigration were significantly increased by myr -PKCε+ ( n = 6) and attenuated with myr- PKCε– ( n = 5) compared to control (n=5) at 120 min (p&lt;0.01). Following SWL, all treatments except myr -PKCε+ with BH 2 significantly reduced H 2 O 2 ( n = 5, p &lt; .01) and increased NO levels ( n = 5, p &lt; .01) compared to control. NO and H 2 O 2 levels following administration of myr -PKCε+ with BH 2 were similar to SWL control. Results suggest that myr -PKCε– attenuates uncoupled eNOS activity and H 2 O 2 release in rat renal veins, after SWL. Promoting coupled eNOS activity with BH 4 yields similar results. Promoting eNOS coupling with BH 4 or inhibiting uncoupled eNOS with myr- PKCε– attenuates oxidative stress and endothelial dysfunction following SWL and BH 2 -induced inflammation.

High Endothelial Venules: A Vascular Perspective on Tertiary Lymphoid Structures in Cancer.

High endothelial venules (HEVs) are specialized postcapillary venules composed of cuboidal blood endothelial cells that express high levels of sulfated sialomucins to bind L-Selectin/CD62L on lymphocytes, thereby facilitating their transmigration from the blood into the lymph nodes (LN) and other secondary lymphoid organs (SLO). HEVs have also been identified in human and murine tumors in predominantly CD3+T cell-enriched areas with fewer CD20+B-cell aggregates that are reminiscent of tertiary lymphoid-like structures (TLS). While HEV/TLS areas in human tumors are predominantly associated with increased survival, tumoral HEVs (TU-HEV) in mice have shown to foster lymphocyte-enriched immune centers and boost an immune response combined with different immunotherapies. Here, we discuss the current insight into TU-HEV formation, function, and regulation in tumors and elaborate on the functional implication, opportunities, and challenges of TU-HEV formation for cancer immunotherapy.

Peripubertal nevoid telangiectasias.

Primary nevoid telangiectasias are a rare, congenital or acquired condition characterized by unilateral telangiectasias with segmental distribution. Acquired nevoid telangiectasias (ANT) are rare – 85 cases in the literature in 2011 –, they prevail in women and the average age at onset is 28 years; the most affected sites are the face, neck and upper chest (6). The prevalence in women, their appearance after taking contraceptives, during pregnancy or, as in the present case at puberty, and their possible regression at the end of pregnancy or in the puerperium, suggest a role for estrogens (3). The congenital forms and the nevoid distribution suggest a mosaic condition that would be made evident by estrogenic stimuli, similarly to what happens for Becker’s nevus, which is sensitive to androgens (2). Histologically, ANT show a dilation of capillaries and post-capillary venules which are lined with normal endothelial cells in the absence of proliferative aspects (2). From the clinical point of view, ANT are unilateral red spots, linear or in small patches, sometimes surrounded by an ischemic halo – Bier’s spots (4) –, which disappear on finger pressure. ANT must be differentiated from acquired port-wine stain (APWS), another rare condition that differs from the much more frequent congenital form for the age of onset. APWS differs from ANT mainly in morphology as it consists of port-wine patches of uniform color similar to the much more frequent congenital ones. The differential diagnosis between acquired ANT and APWS is not always easy, because clinical features and histology can be superimposable (2). Primary ANT should then be differentiated from secondary ANT. The latter are mainly due to hyperestrogenism from liver disease or other diseases (5). The therapy is based on the use of the dye laser (1), but relapses have been reported after this therapy (2).

Post-capillary venules are the key locus for transcytosis-mediated brain delivery of therapeutic nanoparticles

Effective treatments of neurodegenerative diseases require drugs to be actively transported across the blood-brain barrier (BBB). However, nanoparticle drug carriers explored for this purpose show negligible brain uptake, and the lack of basic understanding of nanoparticle-BBB interactions underlies many translational failures. Here, using two-photon microscopy in mice, we characterize the receptor-mediated transcytosis of nanoparticles at all steps of delivery to the brain in vivo. We show that transferrin receptor-targeted liposome nanoparticles are sequestered by the endothelium at capillaries and venules, but not at arterioles. The nanoparticles move unobstructed within endothelium, but transcytosis-mediated brain entry occurs mainly at post-capillary venules, and is negligible in capillaries. The vascular location of nanoparticle brain entry corresponds to the presence of perivascular space, which facilitates nanoparticle movement after transcytosis. Thus, post-capillary venules are the point-of-least resistance at the BBB, and compared to capillaries, provide a more feasible route for nanoparticle drug carriers into the brain.

Abstract 2699: Transcriptome analysis identifies molecular markers of tumor-associated high endothelial venules that predict breast cancer survival

Abstract High endothelial venules (HEVs) are specialized post-capillary venules that recruit naïve lymphocytes to the lymph nodes and are essential for the development of adaptive immunity. HEVs can develop in tumors, and these specialized tumor blood vessels are thought to be important for recruiting naïve T cells and B cells into tumors and locally enhance anti-tumor immunity by promoting tertiary lymphoid structure formation. At present, the genes involved in the development and function of tumor-associated HEVs remain largely unknown. By a comparative transcriptome analysis in human breast cancer, we identified the expression of genes that represents the molecular signature of tumor-associated HEVs distinct from the rest of the tumor vasculature. Tumor vessels highly expressing these genes were associated with the accumulation of T and B lymphocytes and the occurrence of tertiary lymphoid structures, which is known to predict therapeutic response to immune checkpoint inhibitor therapies. Moreover, the Cancer Genome Atlas (TCGA) data show that the high expression of these genes is associated with a significant survival benefit in advanced breast cancer. The molecular signature of HEVs identified here may be useful for guiding immunotherapies and provide a new direction for investigating tumor-associated HEVs and their clinical significance. Citation Format: Junko Sawada, Nobuyoshi Hiraoka, Ashley Fournier-Goss, Masanobu Komatsu. Transcriptome analysis identifies molecular markers of tumor-associated high endothelial venules that predict breast cancer survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2699.

The Clinical-Stage sGC Stimulator IW-1701 Prevents Increase of Plasma Biomarkers of Intravascular Inflammation and Suppresses Leukocyte-Endothelial Interactions in Tnfα-Treated Mice

Soluble guanylate cyclase (sGC) is a heme containing signaling enzyme that catalyzes the formation of cyclic guanosine 3‘,5‘-monophosphate (cGMP) from GTP in response to nitric oxide (NO) binding to heme. The second messenger cGMP modulates many physiological processes including inflammation. Treatment with NO donors or inhibitors of cGMP-selective phosphodiesterase 9 (PDE9) significantly attenuates TNFα-induced intravascular inflammation in C57BL/6 and sickle cell mice. The anti-inflammatory effect of PDE9 inhibitors was augmented by co-administration of hydroxyurea (HU), which is the current standard of care for sickle cell disease. It has been proposed that HU may exert its anti-inflammatory effect by acting as an NO donor. sGC stimulators are small molecules that bind to sGC and enhance NO-dependent catalytic activity of the enzyme. Here we evaluated the effect of the sGC stimulator IW-1701 in the absence and presence of HU on biomarkers of intravascular inflammation, leukocyte-endothelial cell interactions and neutrophil trafficking in C57BL/6 mice.Treatment of C57BL/6 mice with TNFα (50 ng/mouse, i.p.) increased the plasma levels of biomarkers of endothelial cell activation (sP-selectin, sE-selectin, sICAM-1) and leukocyte activation (MIP-2, sL-selectin). Pretreatment of mice with IW-1701 (10 mg/kg, p.o.) reduced the increase in plasma concentrations of MIP-2 and sL-selectin following the TNFα challenge by 81% and 58%, respectively. The TNFα-induced increase in plasma levels of sP-selectin, sE-selectin and sICAM-1 were also inhibited by 31%, 37% and 34%, respectively in IW-1701-treated mice. Co-treatment with HU augmented the effect of IW-1701 on plasma concentrations of sE-selectin and sICAM-1 in TNFα treated mice.The effects of IW-1701, HU and the combination on leukocyte endothelial cell interactions was assessed using intravital microscopy of post-capillary venules of the mouse cremaster muscle. TNFα treatment of C57BL/6 mice decreased leukocyte velocity from 26.6±3.1 μm/sec to 5.5±0.7 μm/sec and leukocyte rolling flux from 43.3±7.1 cells/min to 16.1±2.7 cells/min as compared to control mice. Pre-treatment of mice with IW-1701 (10 mg/kg) alone increased leukocyte velocity and rolling flux to 10.3±1.1 μm/sec and 24.1±2.3 cells/min, respectively. Similarly, pre-treatment of mice with HU (100 mg/kg, p.o.) alone increased leukocyte velocity to 15.5±1.7 μm/sec and leukocyte rolling flux to 50.2±5.9 cells/min. Co-administration of IW-1701 in combination with HU further increased leukocyte velocity (19.7±1.9 μm/sec) and leukocyte rolling flux (64.5±5.5 cells/min) in post-capillary venules of the mouse cremaster muscle. Treatment with either IW-1701, HU, or the combination did not change the number of adherent leukocytes to the vascular wall following TNFα stimulation. To further evaluate the effect of IW-1701 on neutrophil extravasation and trafficking we used a peritoneal recruitment model. Intraperitoneal injection of TNFα induced accumulation of Gr.1 positive neutrophils in the mouse peritoneal cavity (4±0.4×105 PMNs/peritoneal lavage). Trafficking of PMNs into mouse peritoneum was attenuated by treatment of mice with HU (2.7±0.5×105 PMNs/peritoneal lavage). Co-administration of HU and IW-1701 further decreased the number of neutrophils extravasated into the peritoneum of TNFα-challenged mice (1.4±0.3×105 PMNs/peritoneal lavage).In summary, prophylactic treatment with IW-1701 inhibited the TNFα-induced increase in plasma levels of biomarkers of intravascular inflammation and attenuated the effect of TNFα on transient adhesive interactions between leukocytes and endothelial cells in mice. Co-administration of HU significantly reduced neutrophil trafficking and augmented the effect of IW-1701 on biomarkers of endothelial cell activation and transient adhesive interactions in the mouse cremaster muscle. This study supports further evaluation of the sGC stimulator IW-1701 to reduce complications associated with intravascular inflammation such as vaso-occlusive crisis in patients with sickle cell disease. DisclosuresTchernychev:Ironwood Pharmaceuticals: Employment, Other: own stock options. Feil:Ironwood Pharmaceuticals: Other: This research was sponsored by Ironwood Pharmaceuticals. Germano:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Warren:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Lonie:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Feil:Ironwood Pharmaceuticals: Other: This research was sponsored by Ironwood Pharmaceuticals. Milne:Ironwood Pharmaceuticals: Employment, Equity Ownership. Hadcock:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Chien:Ironwood Pharmaceuticals: Employment, Equity Ownership. Currie:Ironwood Pharmaceuticals: Employment, Equity Ownership. Graul:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals .

Changes in Morphometric Indicators of Blood Vessels of Masticatory Muscles in Simulation of Diabetic Angiopathy in Rats

Summary. Relevance. Diabetic joint damage is common in the clinic. The blood vessels of the masticatory muscles that affect the morphogenesis of diabetic arthropathy of the temporomandibular joint remain poorly studied in this pathology.&#x0D; Objective: to study the features of remodeling of blood vessels of the masticatory muscles of the temporomandibular joint in experimental hyperglycemia by quantitative morphological methods.&#x0D; Materials and Methods. The study material was 45 laboratory mature white male rats, which were divided into 3 groups. The 1st group consisted of 15 intact apparently healthy animals, the 2nd included 15 rats with one-month diabetic angiopathy, and the 3rd had 15 rats with two-month specified pathology. Diabetes mellitus was simulated by a single intraperitoneal injection of streptozotocin from Sigma at a dose of 50 mg/kg. Euthanasia of animals was performed by bloodletting under thiopental anesthesia one and two months after the start of the experiment. The blood glucose concentration was determined. Histological micropreparations were made from the temporomandibular joint and the temporal and external pterygoid muscles. Morphometric measurements of small caliber arteries (outer diameter 26-50 μm), veins and microvessels of the hemomicrocirculatory tract were performed on muscle micropreparations; relative volumes of damaged synoviocytes and chondrocytes were determined on micropreparations of joints. Quantitative indicators were processed statistically.&#x0D; Results and Conclusions. It was revealed that the gleam of arteries considerably decreases, their wall thickens, and endotheliocytes are damaged in the studied muscles with the modeled pathology. Venous vessels are dilated, full-blooded with a reduced wall thickness. The most pronounced changes in these experimental conditions are the structure of microvessels, which is characterized by narrowing of arterioles, precapillary arterioles and hemocapillaries, pronounced expansion of postcapillary venules and venules. Plethora, stasis, thrombosis, irregularity of the lumen, sacculation, and varicose expansions were noted in venous microvessels. Venous fullness led to hypoxia, disorders of trophism and metabolism, dystrophy, necrobiosis of muscle fibers and stromal structures, foci of infiltration and sclerosis. It was revealed that morphological changes in the vascular bed of the masticatory muscles affect the morphogenesis of diabetic arthropathy of the temporomandibular joint.

C81-evoked inhibition of the TNFR1-NFκB pathway during inflammatory processes for stabilization of the impaired vascular endothelial barrier for leukocytes.

Chronic inflammation-related diseases are characterized by persistent leukocyte infiltration into the underlying tissue. The vascular endothelium plays a major role in this pathophysiological condition. Only few therapeutic strategies focus on the vascular endothelium as a major target for an anti-inflammatory approach. In this study, we present the natural compound-derived carbazole derivative C81 as chemical modulator interfering with leukocyte-endothelial cell interactions. An in vivo assay employing intravital microscopy to monitor leukocyte trafficking after C81 treatment in postcapillary venules of a murine cremaster muscle was performed. Moreover, in vitro assays using HUVECs and monocytes were implemented. The impact of C81 on cell adhesion molecules and the NFκB signaling cascade was analyzed in vitro in endothelial cells. Effects of C81 on protein translation were determined by incorporation of a puromycin analog-based approach and polysome profiling. We found that C81 significantly reduced TNF-activated leukocyte trafficking in postcapillary venules. Similar results were obtained in vitro when C81 reduced leukocyte-endothelial cell interactions by down-regulating cell adhesion molecules. Focusing on the NFκB signaling cascade, we found that C81 reduced the activation on multiple levels of the cascade through promoted IκBα recovery by attenuation of IκBα ubiquitination and through reduced protein levels of TNFR1 caused by protein translation inhibition. We suggest that C81 might represent a promising lead compound for interfering with inflammation-related processes in endothelial cells by down-regulation of IκBα ubiquitination on the one hand and inhibition of translation on the other hand without exerting cytotoxic effects.

Genesis of Antibiotic Resistance (AR) LXXV: Turbulence Modeling of Simplified Severe Sepsis Protocol‐2 (SSSP‐2), NCT01663701: Simulation of Contributing factors that Protract &amp; Métier Torque Thrusted LPE Aggregates’ in Coandă effect Cause Critical Closing Pressure (CCP)

Hypothesis: EGDT-ERP is directly inducing blood volume expansion, altering the BP, alter the velocity, shear stress, hematocrit forming an LPE aggregate’ and an adverse pressure gradient in vivo smite NFP lead to CCP subsequent CVA/comatose. An effort is made to test a unifying (holistic) hypothesis that has the potential to lull LPE aggregates, alongside AR clinical persisters in the cerebrovascular capillary bed with intrinsic torque of LPE (bluff body) in tandem dragging on the capillary lumen, alongside LE (large eddies), vortices would rupture true capillaries eliciting CCP in an asymmetric velocity profile through anisotropic mode with the collapse of NFP is tested with fluid flow. Geometry: We take inspiration from the impact of torque-drag of a moving object in time sequence: a: torque- and drag sustain damage to the runway – correlate LPR torque – drag on the capillary bed: https://www.youtube.com/watch?v=DFOynlRYT54YouTube©– (time scale: seconds)– b: https://www.youtube.com/watch?v=u5axFVRdNRU; c:splash on the landing: https://www.youtube.com/watch?v=9poWj5eu5rE- (time scale: seconds) – d:https://www.youtube.com/watch?v=2u7jY3V-GJ0– rejected take-off – LPE aggregates lead abrupt occlusion; Governing equations: Darcy's law: Q=kA∆P/μl, -Eq3.27, where Q=discharge flow rate, k=permeability, A= cross-section of the media (blood), ∆P=pressure drop, μ=viscosity, L=length, derived eq. 3.28 – 3.34; (p104-105, ISBN 9780128024089); Torque: τ = r x F, where force F, that act on a particle at position, r(p250 eq;7.6 (ISBN 9780521198110). Experimental Observation: The following experimental observations is an archetype for our hypothesis ad hoc momento. PMID:27319318: Fig2 b, d, f, h compares to the Fig 1, III and V, E, would be lysed passing through the occlusion, releasing lysate, in turn, activating L, P, and E forming LPE; Fig 3d-F represent an LPE aggregate in vivo of sepsis cohort in NCT01663701; Fig 4c the stenosed vessels energy level declined; Fig 5c location II, high energy location VI favor LPE aggregate upstream causing CCP; penultimate to losing NFP, CVA cloture comatose. PMID: 31297646: indicate that RBC(E) aggregate occurs upstream of stenosis forming thin cell free layer (CFL) and decreased flow velocity are in favor of our hypothesis. PMID 31010931 Fig 5 C in the CLP group continuous accumulation of heparin sulfate in the hippocampus of sepsis model (PMID 31297646, PMID 29195076), Fig 7E, implicit of LPE aggregate at the arterial end of the capillary bed;p9213 reinternalize cecum–a trauma-lack≡DDx!; ( PMID: 30720464 PMID: 31337421; PMID: 31201739), patient-specific, TM-CFD; Simulation: Our effort to simulate for water/sewage flow system indicate that a. monitors the aggregate formation, b. utilizes the degreaser/blood-thinning agent, c. coating the pipe agents to repair glycocalyx-endothelium to restore the structure and laminar flow. At present, attempting the simulation of role of CFL in eliciting the adverse pressure gradient / vasomotion in the terminal arteriole - exchange vessels - postcapillary venule in vivo.

A genomic address code directs assembly and function of NKX2‐3:COUP‐TFII complexes that drive organotypic expression of the mucosal vascular addressin

The mucosal vascular addressin cell-adhesion molecule 1 (MAdCAM1) is selectively displayed by intestinal postcapillary and high endothelial venule sites of immune cell recruitment from the blood. It serves as a gatekeeper for lymphocyte entry and is a therapeutic target for inflammatory bowel disease, yet the molecular mechanisms underlying its targeted expression remain unclear. We describe a phylogenetically conserved genomic element within the Madcam1 promoter that integrates intestinal (homeodomain protein Nkx2-3), capillary/arterial (repressive effector proteins of the Notch signaling pathway), and venule-selective (COUP-TFII) transcription regulatory mechanisms with inflammatory (NfkB-dependent) signals to direct MAdCAM1 expression. The regulatory motif cooperatively binds Nkx2-3 and COUP-TFII to activate transcription from the Madcam1 promoter. Heterodimer formation is DNA dependent and is mediated by the Nkx2-3 tinman domain. The Notch downstream repressor Hey1 competes for binding of NKX2-3, and Notch signaling and Hey1 overexpression suppress promoter activity and Madcam1 induction in endothelial cells. Pan-endothelial overexpression of COUP-TFII counters Hey1 repression and drives ectopic MAdCAM1 in Nkx2-3+ intestinal capillaries. The results define a genomic address code for MAdCAM1 expression and control of lymphocyte homing via intestinal venules. Finally, we identify similar composite elements in genes involved in cardiovascular and neuronal development, suggesting a mechanism for combinatorial regulation of organogenesis by COUP-TFII and NXK2 family members.

Integrating Microfluidic Models with Hemoglobin‐Based Oxygen Carriers for Investigating Oxygen‐Mediated Endothelial Function

Inadequate oxygen delivery to tissue, or hypoxia, is caused by extensive abnormalities in the structure and function of the vascular network. Hypoxia also has a significant role in vascular diseases such as stroke, pregnancy disorders, and cancer. Despite its prevalence, there is no established method for alleviating hypoxia and its deleterious effects to vascular function. Existing experimental models are incapable of enabling cellular and intercellular microcirculatory measurements while also controlling the local oxygen environment, impeding our ability to mechanistically study these effects. Hemoglobin-based oxygen carriers (HBOCs) are a promising class of red blood cell substitute which have been well characterized for transfusion medicine applications, yet little has been done to study the effects of HBOC administration on vascular function at the cellular level. In this work, we put forth a novel integration of microfluidic vascular models with HBOCs to investigate the effects of oxygen transport in microcirculation in vitro. We hypothesize that alleviating hypoxia with HBOCs restores normal blood vessel structure and function. We fabricated fully-lumenized microvessels in vitro which mimic post-capillary venules. We assessed vessel stability by immunofluorescence staining, and permeability under environmentally-controlled normoxic and hypoxic conditions. We then synthesized polymerized hemoglobin (PolyHb), an HBOC with favorable molecular weight and oxygen-carrying properties to alleviate hypoxia while minimizing extravasation and subsequent toxicity. We validated that PolyHb may be synthesized under varying conditions to selectively control the HBOC size and oxygen affinity, demonstrating that these properties can be modified to enable favorable oxygen transport and may be varied based on the desired application. As compared to previous generation HBOCs, PolyHb can attain molecular weights which will enable intravascular circulation to minimize toxicity, making PolyHb an ideal candidate for microfluidic investigation into the physiological effects of rescuing hypoxia. In future work, we will perfuse PolyHb through the engineered microvessels at constant flow and physiological shear stress levels to assess the ability of PolyHb to relieve induced hypoxia under imposed circumferential and longitudinally oriented hypoxia gradients.

The enigma of adult onset Henoch Schoenlein purpura and leucocytoclastic Vasculits: a case report

&lt;p class="abstract"&gt;Leukocytoclastic vasculitis (LCV), also known as hypersensitivity vasculitis is a small vessel inflammatory disease which mainly involves the postcapillary venules. It can be idiopathic or secondary to connective tissue diseases, infection, malignancy. A 39 year old male presented with a recurrent episode of multiple palpable purpurae over the legs, trunk, back and upper limbs with diffuse abdominal pain, bloody diarrhoea and arthralgia. Anti-nuclear antibodies (ANA) were positive, but ANA profile was equivocal. Histopathological examination confirmed leukocytoclastic vasculitis. A final diagnosis of Henoch Schoenlein purpura (HSP) was reached based on EULAR Criteria. He was treated with tapering doses of prednisolone, hydroxychloroquine and bed rest with limb elevation. All symptoms and lesions gradually resolved. Various sub-types of cutaneous small vessel vasculitis may mimic each other, warranting a meticulous evaluation. Adult onset HSP though rare must be considered in the differential diagnosis of LCV, which might be the presenting manifestation of an underlying connective tissue disease. Hence long-term follow-up with stringent clinical and laboratory monitoring is recommended.&lt;/p&gt;

599 Single-cell transcriptome profiling reveals vascular endothelial cell heterogeneity in human skin

Vascular endothelial cells (ECs) are increasingly recognized as active players in intercellular crosstalk more than passive linings of a conduit for nutrition delivery. Yet, their functional roles and heterogeneity in skin remain uncharacterized. To investigate the tissue-specific features and intra-tissue heterogeneity in dermal ECs, human dermal EC atlas of over 23,000 single-cell transcriptomes was obtained and further analyzed. In comparison with ECs from other human tissues, extracted from previously reported data, dermal ECs possess unique characteristics in transcriptional modulation, metabolic and chemotactic functions. We identified five major subtypes of human dermal ECs and their molecular signatures. Within different layers in dermis, ECs are also varied in their biological activities. Metabolic transcriptome analysis revealed a gradual switch from oxidative phosphorylation to glycolysis in dermal EC subtypes along the blood flow. Moreover, dermal capillary ECs abundantly expressed HLA class II molecules, suggesting its immune-surveillance role. ECs in post-capillary venules, with high levels of adhesion molecules, were equipped with possible capacity in immune cell arrest, adhesion, and infiltration. Our study provides a comprehensive characterization of EC features and heterogeneity in human dermis and sets the stage for future research in identifying disease-specific alterations of dermal ECs in various dermatoses.

Noninvasive diagnostic techniques of port wine stain.

Port-wine stain (PWS) is a benign capillary malformation that most commonly occurs in the head and neck. It is present at birth and progresses over time. It is formed by progressive dilatation of post-capillary venules and is associated with hypertrophy and nodularity with increasing age, leading to cosmetic disfigurement and psychological aggravation. It is caused by genetic mosaicism in GNAQ and GNA11 genes. Histopathology is the gold standard for assessment of PWS but it is invasive and may cause scarring. Inadequate characterization of the lesions may predispose to inadequate treatment protocols as well as higher treatment dosages. Clinical evaluation of treatment efficacy is subjective and may not be a representative of actual results. Therefore, an objective visualization modality is required. With evolving technology, numerous optical instruments have been developed for objective evaluation and visualization of subsurface structures. These include VISIA-CR™ system, videodermoscopy, high-frequency ultrasound (HFUS), laser speckle contrast imaging (LSCI), reflectance spectrophotometers and tristimulus colorimeter, laser Doppler flowmetry (LDF), cross-polarized diffuse reflectance imaging system (CDR), reflectance confocal microscopy (RCM), optical coherence tomography (OCT), and spatial frequency domain imaging (SFDI). These semi-quantitative modes of diagnosis are complementary to each other. Some can be used in the clinical setting while others, due to high instrument cost, are limited to the research settings. In this review, we bring to you a brief overview of noninvasive diagnostic modalities in PWS.

Pathophysiology of light phenotype SARS-CoV-2 interstitial pneumonia: from histopathological features to clinical presentations

Little is known about the light phenotype of SARS-CoV-2 pneumonia, which behaves in an unusual way, unlike other known respiratory diseases. We believe that the histopathological features of early COVID-19 could be considered the pathophysiological hallmark of this disease.Lung cryobiopsies show almost pristine alveoli, enlarged/hyperplasic alveolar capillaries along with dilatation of the post capillary pulmonary venules. Hypoxemia could therefore be explained by a reduction of the normal V/Q ratio, due to blood overflow around well ventilated alveoli.This could clarify typical manifestations of type L COVID-19, such as happy hypoxemia, response to awake prone positioning, response to PEEP/CPAP and platypnea orthodeoxia.

The capillary fascicle in skeletal muscle: Structural and functional physiology of RBC distribution in capillary networks.

The capillary module, consisting of parallel capillaries from arteriole to venule, is classically considered as the building block of complex capillary networks. In skeletal muscle, this structure fails to address how blood flow is regulated along the entire length of the synchronously contracting muscle fibres. Using intravital video microscopy of resting extensor digitorum longus muscle in rats, we demonstrated the capillary fascicle as a series of interconnected modules forming continuous columns that align naturally with the dimensions of the muscle fascicle. We observed structural heterogeneity for module topology, and functional heterogeneity in space and time for capillary-red blood cell (RBC) haemodynamics within a module and between modules. We found that module RBC haemodynamics were independent of module resistance, providing direct evidence for microvascular flow regulation at the level of the capillary module. The capillary fascicle is an updated paradigm for characterizing blood flow and RBC distribution in skeletal muscle capillary networks. Capillary networks are the fundamental site of oxygen exchange in the microcirculation. The capillary module (CM), consisting of parallel capillaries from terminal arteriole (TA) to post-capillary venule (PCV), is classically considered as the building block of complex capillary networks. In skeletal muscle, this structure fails to address how blood flow is regulated along the entire length of the synchronously contracting muscle fibres, requiring co-ordination from numerous modules. It has previously been recognized that TAs and PCVs interact with multiple CMs, creating interconnected networks. Using label-free intravital video microscopy of resting extensor digitorum longus muscle in rats, we found that these networks form continuous columns of linked CMs spanning thousands of microns, herein denoted as the capillary fascicle (CF); this structure aligns naturally with the dimensions of the muscle fascicle. We measured capillary-red blood cell (RBC) haemodynamics and module topology (n=9 networks, 327modules, 1491 capillary segments). The average module had length 481μm, width 157μm and 9.51parallel capillaries. We observed structural heterogeneity for CM topology, and functional heterogeneity in space and time for capillary-RBC haemodynamics within a module and between modules. There was no correlation between capillary RBC velocity and lineal density. A passive inverse relationship between module length and haemodynamics was remarkably absent, providing direct evidence for microvascular flow regulation at the level of the CM. In summary, the CF is an updated paradigm for characterizing RBC distribution in skeletal muscle, and strengthens the theory of capillary networks as major contributors to the signal that regulates capillary perfusion.

From SARS-CoV-2 hematogenous spreading to endothelial dysfunction: clinical-histopathological study of cutaneous signs of COVID-19

BackgroundTo date, very few studies on clinical-histopathological correlations of cutaneous disorders associated with COVID-19 have been conducted.Case presentationThe Case 1 was a 90-year-old man, who tested positive for SARS-CoV-2 from a nasopharyngeal swab. Two days later, he was hospitalized and after eleven days transferred to Intensive Care Unit. A chest CT showed bilateral ground-glass opacities. Just that day, an erythematous maculo-papular rash appeared on trunk, shoulders and neck, becoming purpuric after few days. Histological evaluations revealed a chronic superficial dermatitis with purpuric aspects. The superficial and papillary dermis appeared edematous, with a perivascular lympho-granulocytic infiltrate and erythrocytic extravasation. At intraepithelial level, spongiosis and a granulocyte infiltrate were detected. Arterioles, capillaries and post-capillary venules showed endothelial swelling and appeared ectatic. The patient was treated with hydroxychloroquine, azithromycin, lopinavir-ritonavir and tocilizumab. Regrettably, due to severe lung impairment, he died.The Case 2 was a 85-year-old man, admitted to Intensive Care Unit, where he was intubated. He had tested positive for SARS-CoV-2 from a nasopharyngeal swab two days before. A chest RX showed bilateral atypical pneumonia. After seven days, a cutaneous reddening involving trunk, upper limbs, neck and face developed, configuring a sub-erythroderma. Histological evaluations displayed edema in the papillary and superficial reticular dermis, and a perivascular lymphocytic infiltrate in the superficial dermis. The patient was treated with hydroxychloroquine, azithromycin, lopinavir-ritonavir and tocilizumab. Sub-erythroderma as well as respiratory symptoms gradually improved until healing.ConclusionsThe endothelial swelling detected in the Case 1 could be a morphological expression of SARS-CoV-2-induced endothelial dysfunction. We hypothesize that cutaneous damage could be initiated by endothelial dysfunction, caused by SARS-CoV-2 infection of endothelial cells or induced by immune system activation. The disruption of endothelial integrity could enhance microvascular permeability, extravasation of inflammatory cells and cytokines, with cutaneous injury. The Case 2 developed a sub-erythroderma associated with COVID-19, and a non-specific chronic dermatitis was detected at histological level. We speculate that a purpuric rash could represent the cutaneous sign of a more severe coagulopathy, as highlighted histologically by vascular abnormalities, while a sub-erythroderma could be expression of viral hematogenous spreading, inducing a non-specific chronic dermatitis.