Intact Microvessels Research Articles

Impact of Matrix Gel Variations on Primary Culture of Microvascular Endothelial Cell Function.

The endothelium regulates crucial aspects of vascular function, including hemostasis, vasomotor tone, proliferation, immune cell adhesion, and microvascular permeability. Endothelial cells (ECs), especially in arterioles, are pivotal for flow distribution and peripheral resistance regulation. Investigating vascular endothelium physiology, particularly in microvascular ECs, demands precise isolation and culturing techniques. Freshly isolated ECs are vital for examining protein expression, ion channel behavior, and calcium dynamics. Establishing primary endothelial cell cultures is crucial for unraveling vascular functions and understanding intact microvessel endothelium roles. Despite the significance, detailed protocols and comparisons with intact vessels are scarce in microvascular research. We developed a reproducible method to isolate microvascular ECs, assessing substrate influence by cultivating cells on fibronectin and gelatin matrix gels. This comparative approach enhances our understanding of microvascular endothelial cell biology. Microvascular mesenteric ECs expressed key markers (VE-cadherin and eNOS) in both matrix gels, confirming cell culture purity. Under uncoated conditions, ECs were undetected, whereas proteins linked to smooth muscle cells and fibroblasts were evident. Examining endothelial cell (EC) physiological dynamics on distinct matrix substrates revealed comparable cell length, shape, and Ca2+ elevations in both male and female ECs on gelatin and fibronectin matrix gels. Gelatin-cultured ECs exhibited analogous membrane potential responses to acetylcholine (ACh) or adenosine triphosphate (ATP), contrasting with their fibronectin-cultured counterparts. In the absence of stimulation, fibronectin-cultured ECs displayed a more depolarized resting membrane potential than gelatin-cultured ECs. Gelatin-cultured ECs demonstrated electrical behaviors akin to intact endothelium from mouse mesenteric arteries, thus advancing our understanding of endothelial cell behavior within diverse microenvironments.

Isolated Fragments of Intact Microvessels: Tissue Vascularization, Modeling, and Therapeutics.

The microvasculature is integral to nearly every tissue in the body, providing not only perfusion to and from the tissue, but also homing sites for immune cells, cellular niches for tissue dynamics, and cooperative interactions with other tissue elements. As a microtissue itself, the microvasculature is a composite of multiple cell types exquisitely organized into structures (individual vessel segments and extensive vessel networks) capable of considerable dynamics and plasticity. Consequently, it has been challenging to include a functional microvasculature in assembled or fabricated tissues. Isolated fragments of intact microvessels, which retain the cellular composition and structures of native microvessels, are proving effective in a variety of vascularization applications including tissue invitro disease modeling, vascular biology, mechanistic discovery, and tissue prevascularization in regenerative therapeutics and grafting. In this review, we will discuss the importance of recapitulating native tissue biology and the successful vascularization applications of isolated microvessels.

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments.

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for the design of personalized microtissues for health screening and drug testing. Current strategies are often quite complex and fail to accurately fully depict the multicellular and functional properties of thermogenic adipose tissue. Microvascular fragments, small intact microvessels comprised of arteriole, venules, and capillaries isolated from adipose tissue, serve as a single autologous source of cells that enable vascularization and adipose tissue formation. This article describes methods for optimizing culture conditions to enable the generation of three-dimensional, vascularized, and functional thermogenic adipose tissues from microvascular fragments, including protocols for isolating microvascular fragments from adipose tissue and culture conditions. Additionally, best practices are discussed, as are techniques for characterizing the engineered tissues, and sample results from both rodent and human microvascular fragments are provided. This approach has the potential to be utilized for the understanding and development of treatments for obesity and metabolic disease.

Atomic Force Microscopy-Based Measurements of Retinal Microvessel Stiffness in Mice with Endothelial-Specific Deletion of CCN1.

Vascular stiffness is an independent predictor of human vascular diseases and is linked to ischemia, diabetes, high blood pressure, hyperlipidemia, and/or aging. Blood vessel stiffening increases owing to changes in the microscale architecture and/or content of extracellular, cytoskeletal, and nuclear matrix proteins. These alterations, while best appreciated in large blood vessels, also gradually occur in the microvasculature and play an important role in the initiation and progression of numerous microangiopathies including diabetic retinopathy. Although macroscopic measurements of arterial stiffness by pulse wave velocity are often used for clinical diagnosis, stiffness changes of intact microvessels and their causative factors have not been characterized. Herein, we describe the use of atomic force microscopy (AFM) to determine stiffness of mouse retinal capillaries and assess its regulation by the cellular communication network (CCN) 1, a stiffness-sensitive gene-encoded matricellular protein. AFM yields reproducible measurements of retinal capillary stiffness in lightly fixed freshly isolated retinal flat mounts. AFM measurements also show significant changes in compliance properties of the retinal microvasculature of mice with endothelial-specific deletion of CCN1, indicating that CCN1 expression, or lack thereof, affects the mechanical properties of microvascular cells in vivo. Thus, AFM has the force sensitivity and the spatial resolution necessary to measure the local modulus of retinal capillaries in situ and eventually to investigate microvascular compliance heterogeneities as key components of disease pathogenesis.

Proteomic Analysis of Control and Disrupted Endothelial Monolayers

The microvascular wall regulates normal blood‐tissue exchange, but under inflammatory conditions, elevated endothelial permeability leads to edema, poor tissue oxygenation, and disrupted tissue homeostasis. Much remains unknown about the mechanisms involved. Using an unbiased proteomic analysis of confluent human umbilical vein endothelial cell (HUVEC) monolayers that were stimulated with 0.1 U/mL thrombin, which produces a time‐dependent increase in permeability that peaks 30 min and recovers to baseline levels at 120 min, novel candidate proteins that may participate in this mechanism were identified. Cell lysates were collected, proteins digested, and the peptides were characterized using a Thermo Q‐exactive‐HF mass spectrometer coupled to a Thermo Easy nLC 1200 to get label‐free quantitation (LFQ) of relative protein expression. High‐resolution mass spectrometric data were analyzed on the MaxQuant processing suite, and the protein groups file subsequently analyzed with the Perseus processing suite. The analysis revealed identified 4627 unique proteins present in HUVEC. Ratios of the LFQ intensities of 30 min. thrombin vs. control and 120 min. thrombin vs. control were generated. Statistical significance (p < 0.05) for 30 min. thrombin vs. control or 120 min. thrombin vs. control was determined using Welch’s t‐test of log(2)‐transformed LFQ intensities. In addition, a z‐score cutoff calculated as [(t test difference of individual protein) − (median t test difference of dataset)] / (standard deviation of t test difference of dataset) was used. The criteria of |z‐score| > 1 and p < 0.05 with Welch’s t test were utilized to filter the dataset, identifying 214 proteins to have altered expression after 30 min thrombin treatment, and 172 proteins having altered expression after 120 min thrombin. When entering the filtered datasets into String‐db, tight junction signaling was identified as enriched with thrombin treatment. Employing a more stringent filtering approach to identify candidate proteins for further study (|z‐score| > 2), we found that the tight junction protein claudin‐11 (CLDN11) had significantly decreased expression after 30 min thrombin (73% decrease from control) but was not significant changed at 120 min. Expression of CLDN11 was confirmed to be decreased by thrombin at 30 min using SimpleWestern (WES) analysis. To determine whether experimental depletion of CLDN11 expression decreases endothelial barrier function, selective CLDN11 siRNA was used. However, evaluation of barrier function using electric cell‐substrate impedance sensing showed no significant changes for up to 48 h after transfection with the siRNA, when CLDN11 protein levels were significantly decreased. Immunofluorescence labeling of CLDN11 in confluent HUVEC revealed that while some CLDN11 could be found in junctions, most labeling was punctate in the cytoplasm, possibly residing in vesicles. Collectively, the results suggest that thrombin significantly alters endothelial tight junctions and reduces CLDN11 expression. However, selective reduction of CLDN11 levels alone does not appear to be sufficient to alter HUVEC monolayer barrier function, and this may be because most CLDN11 fails to localize in junctions in this cultured endothelial cell model. Future studies will be directed at evaluating CLDN11 localization in postcapillary venules and determining its potential role in microvascular barrier function using intact microvessels and in vivo.

The Evaluation of Neovessel Angiogenesis Behavior at Tissue Interfaces.

Angiogenesis, the formation of new vessel elements from existing vessels, is important in homeostasis and tissue repair. Dysfunctional angiogenesis can contribute to numerous pathologies, including cancer, ischemia, and chronic wounds. In many instances, growing vessels must navigate along or across tissue-associated boundaries and interfaces tissue interfaces. To understand this dynamic, we developed a new model for studying angiogenesis at tissue interfaces utilizing intact microvessel fragments isolated from adipose tissue. Isolated microvessels retain their native structural and cellular complexity. When embedded in a 3D matrix, microvessels, sprout, grow, and connect to form a neovasculature. Here, we discuss and describe methodology for one application of our microvessel-based angiogenesis model, studying neovessel behavior at tissue interfaces.

Cerebrovascular Senescence Is Associated With Tau Pathology in Alzheimer's Disease.

Alzheimer's Disease (AD) is associated with neuropathological changes, including aggregation of tau neurofibrillary tangles (NFTs) and amyloid-beta plaques. Mounting evidence indicates that vascular dysfunction also plays a key role in the pathogenesis and progression of AD, in part through endothelial dysfunction. Based on findings in animal models that tau pathology induces vascular abnormalities and cellular senescence, we hypothesized that tau pathology in the human AD brain leads to vascular senescence. To explore this hypothesis, we isolated intact microvessels from the dorsolateral prefrontal cortex (PFC, BA9) from 16 subjects with advanced Braak stages (Braak V/VI, B3) and 12 control subjects (Braak 0/I/II, B1), and quantified expression of 42 genes associated with senescence, cell adhesion, and various endothelial cell functions. Genes associated with endothelial senescence and leukocyte adhesion, including SERPINE1 (PAI-1), CXCL8 (IL8), CXCL1, CXCL2, ICAM-2, and TIE1, were significantly upregulated in B3 microvessels after adjusting for sex and cerebrovascular pathology. In particular, the senescence-associated secretory phenotype genes SERPINE1 and CXCL8 were upregulated by more than 2-fold in B3 microvessels after adjusting for sex, cerebrovascular pathology, and age at death. Protein quantification data from longitudinal plasma samples for a subset of 13 (n = 9 B3, n = 4 B1) subjects showed no significant differences in plasma senescence or adhesion-associated protein levels, suggesting that these changes were not associated with systemic vascular alterations. Future investigations of senescence biomarkers in both the peripheral and cortical vasculature could further elucidate links between tau pathology and vascular changes in human AD.

Prolonged activation of cAMP signaling leads to endothelial barrier disruption via transcriptional repression of RRAS.

The increase in cAMP levels in endothelial cells triggers cellular signaling to alter vascular permeability. It is generally considered that cAMP signaling stabilizes the endothelial barrier function and reduces permeability. However, previous studies have only examined the permeability shortly after cAMP elevation and thus have only investigated acute responses. Because cAMP is a key regulator of gene expression, elevated cAMP may have a delayed but profound impact on the endothelial permeability by altering the expression of the genes that are vital for the vessel wall stability. The small guanosine triphosphate hydrolase Ras-related protein (R-Ras) stabilizes VE-cadherin clustering and enhances endothelial barrier function, thereby stabilizing the integrity of blood vessel wall. Here we show that cAMP controls endothelial permeability through RRAS gene regulation. The prolonged cAMP elevation transcriptionally repressed RRAS in endothelial cells via a cAMP response element–binding 3 (CREB3)–dependent mechanism and significantly disrupted the adherens junction. These effects resulted in a marked increase of endothelial permeability that was reversed by R-Ras transduction. Furthermore, cAMP elevation in the endothelium by prostaglandin E2 or phosphodiesterase type 4 inhibition caused plasma leakage from intact microvessels in mouse skin. Our study demonstrated that, contrary to the widely accepted notion, cAMP elevation in endothelial cells ultimately increases vascular permeability, and the cAMP-dependent RRAS repression critically contributes to this effect.—Perrot, C. Y., Sawada, J., Komatsu, M. Prolonged activation of cAMP signaling leads to endothelial barrier disruption via transcriptional repression of RRAS.

Assessing Endothelial Cell Permeability and Transport Pathways via Biotin/FITC‐Avidin Interaction in Cultured Endothelial Microchannel Networks Using Microfluidic Devices

Microfluidic technologies have enabled in vitro studies to closely simulate the in vivo microvessel environment with sufficient complexity. Our recent studies demonstrated that endothelial cells (ECs) cultured under continuous flow within microfluidic devices form junctions closer to those observed in intact microvessels than static cell cultures. Importantly their cell signaling responses to inflammatory mediators are comparable to those derived from individually perfused intact venules. However, the commonly used impermeable polydimethysiloxane (PDMS) in microfluidic devices could not study the permeability properties of ECs. Recently the biotin‐avidin interaction has been successfully applied to static cell cultures for assessing EC permeability. The objective of this study is to apply the biotin‐avidin approach to cultured ECs in microfluidic device, and detect the spatial changes in EC permeability and differentiate transport pathways under control and stimulated conditions. Microfluidic devices were fabricated through soft lithography at different levels of channel branches. Glass coverslips were cohered as the base of devices for image acquisition. Inner channels of device were coated with biotinylated fibronectin before rat primary dermal microvascular endothelial cells were seeded and cultured along the lining of channels with a constant medium flow at 0.22 μL/min. ECs reach confluence within 3 days from seeding and successfully covered the inner surface of microchannels. Permeability was assessed by measuring integrated fluorescence intensity of region of interest after perfusion the microchannel networks with solutions containing FITC‐avidin with confocal images. The results showed that perfusion microchannels with medium alone showed no fluorescent signals. Only scattered FITC signals were observed at cellular junctions when the microchannels were perfused with BSA‐Ringer's solution containing FITC‐avidin (served as control). When PAF (10 nM) was added to the perfusate in the presence of FITC‐avidin, we observed a time dependent increases in fluorescent areas and intensity along junctions between ECs. Image analysis showed that fluorescence intensity increased 6.6 ± 0.12 and 8.94 ± 0.25 times that of the control when PAF was perfused for 4 and 10 min, respectively. The spatial distribution of FITC signal indicates paracellular pathway as the predominant mechanism of PAF‐induced permeability increase. The biotin‐avidin approach is based on high affinity ligand‐ acceptor interactions and their interaction enable the mapping of transport pathways and quantify the leaked molecules. Our study indicated that the application of biotin‐avidin approach to microfluidic devices with cultured EC networks could serve as a useful tool for assessing permeability properties of ECs that were grown under continuous flow, a cell culture condition closer to that in vivo.Support or Funding InformationSupported by HL130363 and DK097391This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reduction of Endothelial Nitric Oxide Increases the Adhesiveness of Constitutive Endothelial Membrane ICAM-1 through Src-Mediated Phosphorylation.

Nitric oxide (NO) is a known anti-adhesive molecule that prevents platelet aggregation and leukocyte adhesion to endothelial cells (ECs). The mechanism has been attributed to its role in the regulation of adhesion molecules on leukocytes and the adhesive properties of platelets. Our previous study conducted in rat venules found that reduction of EC basal NO synthesis caused EC ICAM-1-mediated firm adhesion of leukocytes within 10–30 min. This quick response occurred in the absence of alterations of adhesion molecules on leukocytes and also opposes the classical pattern of ICAM-1-mediated leukocyte adhesion that requires protein synthesis and occurs hours after stimulation. The objective of this study is to investigate the underlying mechanisms of reduced basal NO-induced EC-mediated rapid leukocyte adhesion observed in intact microvessels. The relative levels of ICAM-1 at different cell regions and their activation status were determined with cellular fractionation and western blot using cultured human umbilical vein ECs. ICAM-1 adhesiveness was determined by immunoprecipitation in non-denatured proteins to assess the changes in ICAM-1 binding to its inhibitory antibody, mAb1A29, and antibody against total ICAM-1 with and without NO reduction. The adhesion strength of EC ICAM-1 was assessed by atomic force microscopy (AFM) on live cells. Results showed that reduction of EC basal NO caused by the application of caveolin-1 scaffolding domain (AP-CAV) or NOS inhibitor, L-NMMA, for 30 min significantly increased phosphorylated ICAM-1 and its binding to mAb1A29 in the absence of altered ICAM-1 expression and its distribution at subcellular regions. The Src inhibitor, PP1, inhibited NO reduction-induced increases in ICAM-1 phosphorylation and adhesive binding. AFM detected significant increases in the binding force between AP-CAV-treated ECs and mAb1A29-coated probes. These results demonstrated that reduced EC basal NO lead to a rapid increase in ICAM-1 adhesive binding via Src-mediated phosphorylation without de novo protein synthesis and translocation. This study suggests that a NO-dependent conformational change of constitutive EC membrane ICAM-1 might be the mechanism of rapid ICAM-1 dependent leukocyte adhesion observed in vivo. This new mechanistic insight provides a better understanding of EC/leukocyte interaction-mediated vascular inflammation under many disease conditions that encounter reduced basal NO in the circulation system.

Thermo-sensitive assembly of the biomaterial REP reduces hematoma volume following collagenase-induced intracerebral hemorrhage in rats

Intracerebral hemorrhage (ICH) frequently results in severe disabilities and high mortality. RGD-containing elastin-like polypeptide (REP), a modified elastin-like polypeptide (ELP), is a thermally responsive biopolymer. REP has high affinity for cells and is known to show non-immunotoxicity, -cytotoxicity, and -inflammatory responses. Here we found that administration of REP in the acute phase of the ICH rat model reduced the hematoma volume, decreased the number of activated microglia, attenuated the expression of von Willebrand Factor (vWF), and prevented the leakage of immunoglobulin G (IgG) into the cerebral parenchyma without any occlusion of intact microvessels. Therefore, the present data suggest that REP treatment could be a novel therapeutic strategy for attenuating the acute phase of ICH.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production.

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca(2+)]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca(2+)]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Sphingosine-1-phosphate Maintains Normal Vascular Permeability by Preserving Endothelial Surface Glycocalyx in Intact Microvessels.

S1P was found to protect the ESG by inhibiting MMP activity-dependent shedding of ESG in cultured endothelial cell studies. We aimed to further test that S1P contributes to the maintenance of normal vascular permeability by protecting the ESG in intact microvessels. We quantified the ESG in post-capillary venules of rat mesentery and measured the vascular permeability to albumin in the presence and absence of 1μM S1P. We also measured permeability to albumin in the presence of MMP inhibitors and compared the measured permeability with those predicted by a transport model for the inter-endothelial cleft. We found that in the absence of S1P, the fluorescence intensity of the FITC-anti-HS-labeled ESG was ~10% of that in the presence of S1P, whereas the measured permeability to albumin was~6.5-fold of that in the presence of S1P. Similar results wereobserved with MMP inhibition. The predictions by the mathematical model further confirmed that S1P maintains microvascular permeability by preserving ESG. Our results show that S1P contributes to the maintenance of normal vascular permeability by protecting the ESG in intact microvessels, consistent with parallel observation in cultured endothelial monolayers.

Inhibition of endothelial nitric oxide synthase decreases breast cancer cell MDA-MB-231 adhesion to intact microvessels under physiological flows.

Nitric oxide (NO) at different concentrations may promote or inhibit tumor growth and metastasis under various conditions. To test the hypothesis that tumor cells prefer to adhere to the locations with a higher endothelial NO production in intact microvessels under physiological flows and to further test that inhibiting NO production decreases tumor cell adhesion, we used intravital fluorescence microscopy to measure NO production and tumor cell adhesion in postcapillary venules of rat mesentery under normal and reduced flow conditions, and in the presence of an endothelial nitric oxide synthase (eNOS) inhibitor, N(G)-monomethyl-l-arginine (l-NMMA). Rats (SD, 250-300 g) were anesthetized. A midline incision (∼2 inch) was made in the abdominal wall, and the mesentery was taken out from the abdominal cavity and spread over a coverslip for the measurement. An individual postcapillary venule (35-50 μm) was first loaded with 4,5-diaminofluorescein diacetate (DAF-2 DA), a fluorescent indictor for NO. Then the DAF-2 intensity was measured for 30 min under a normal or reduced flow velocity, with and without perfusion with MDA-MB-231 breast cancer cells, and in the presence of l-NMMA. We found that tumor cells prefer to adhere to the microvessel locations with a higher NO production such as curved portions. Inhibition of eNOS by l-NMMA attenuated the flow-induced NO production and reduced tumor cell adhesion. We also found that l-NMMA treatment for ∼40 min reduced microvessel permeability to albumin. Our results suggest that inhibition of eNOS is a good approach to preventing tumor cell adhesion to intact microvessels under physiological flows.

Circulating microparticles in pancreatic tumor patients and their roles in mediating leukocyte adhesion in intact microvessels

Pancreatic cancer is one of the nation's deadliest cancers with a dismal 6 percent five‐year survival rate. Pancreatic cancer characteristically creates an abnormal inflammatory or desmoplastic response, which may play a role in its resistance to systemic therapy and aggressive metastasis. Recent studies in circulating microparticles (MPs), the small plasma membrane‐derived vesicles (0.1–1um) released during cell activation, indicated that elevated MPs occur in many cardiovascular diseases, as well as in cancer patients. Since the released MPs carry a range of antigens depending on the type and activation state of their parental cells, they have been considered as biomarker to indicate the disease status and predict the prognosis. The objectives of our study are to investigate the relationship between pancreatic cancer and vascular inflammation and evaluate whether circulating MPs can be a potential biomarker for pancreatic cancer prognosis and treatment outcomes. In this study we characterized the profile of circulating MPs in patients with malignant and non‐malignant pancreatic diseases, investigated the interaction of cultured pancreatic cancer cell‐derived MPs (PACMPs) with blood and endothelial cell‐derived MPs, and explored the roles of PACMPs in mediating vascular inflammation. Flow cytometry was used to quantify plasma MPs, and identify MP cell origins using antibodies directly against cell specific antigens. Compared with healthy controls (n=8), patients with pancreatic cancer (n=11) and pancreatic cyst (nonmalignant, n=7) all had elevated levels of MPs. About 90% of total plasma MPs were from platelets, leukocytes, and endothelial cells. Over 50% of the MPs existed as aggregated units expressing multiple parental cell surface antigens. Electron microscopy and atomic force microscopy delineated the aggregation profiles of plasma MPs. When PACMPs were introduced into cancer patients’ plasma, more than 50% of the plasma MPs bound with PACMPs, and 32% of the plasma MPs that bound with PACMPs express platelet specific antigen. The interaction between PACMPs and vascular endothelial cells were investigated in individually perfused rat venules. After perfusing vessels with PACMPs followed by annexin V staining, confocal images show a significant number of adhered PATMPs in the microvessel walls. After resuming blood flow in PACMP perfused vessels, leukocyte adhesion was significantly increased from 3 ± 1 (control) to 13 ± 3 per 100 um of vessel length. Our results showed that both patients with malignant and non‐malignant pancreatic diseases had elevated circulating MPs, indicating an association between pancreatic diseases and inflammation. The increased plasma MPs, mainly from blood cells and ECs origins, and PACMPS are highly adhesive. They not only interact with each other, but also interact with normal ECs and mediate leukocyte adhesion, which may play important roles in propagation of the inflammation and contribute to pancreatic cancer progression and metastasis.Support or Funding InformationSupported by HL056237, HL084338 and DK097391.

In vitro recapitulation of functional microvessels for the study of endothelial shear response, nitric oxide and [Ca2+]i.

Microfluidic technologies enable in vitro studies to closely simulate in vivo microvessel environment with complexity. Such method overcomes certain constrains of the statically cultured endothelial monolayers and enables the cells grow under physiological range of shear flow with geometry similar to microvessels in vivo. However, there are still existing knowledge gaps and lack of convincing evidence to demonstrate and quantify key biological features of the microfluidic microvessels. In this paper, using advanced micromanufacturing and microfluidic technologies, we presented an engineered microvessel model that mimicked the dimensions and network structures of in vivo microvessels with a long-term and continuous perfusion capability, as well as high-resolution and real-time imaging capability. Through direct comparisons with studies conducted in intact microvessels, our results demonstrated that the cultured microvessels formed under perfused conditions recapitulated certain key features of the microvessels in vivo. In particular, primary human umbilical vein endothelial cells were successfully cultured the entire inner surfaces of the microchannel network with well-developed junctions indicated by VE-cadherin staining. The morphological and proliferative responses of endothelial cells to shear stresses were quantified under different flow conditions which was simulated with three-dimensional shear dependent numerical flow model. Furthermore, we successfully measured agonist-induced changes in intracellular Ca2+ concentration and nitric oxide production at individual endothelial cell levels using fluorescence imaging. The results were comparable to those derived from individually perfused intact venules. With in vivo validation of its functionalities, our microfluidic model demonstrates a great potential for biological applications and bridges the gaps between in vitro and in vivo microvascular research.

Abstract T P232: Combination Therapy With Taurine Plus Tissue Plasminogen Activator in a Rat Model of Embolic Stroke

Background: Tissue plasminogen activator (tPA) is the only FDA-approved therapy for acute ischemic stroke, but it has narrow therapeutic window (within 4.5h) and increased risks of intracerebral hemorrhage. Taurine, an endogenous 2-aminoethansulfolic amino acid, exhibits a plethora of physiological functions. It has been found to protect against ischemic brain injury in animal models of stroke by diverse mechanisms. Strikingly, published data show that the therapeutic window of taurine was at least 8h after the onset of ischemic stroke, thus potentially allowing for delayed or more effective thrombolysis. In this study we investigated whether combination therapy with taurine plus tPA can block hemorrhagic transformation and thus extend the time window of tPA after delayed tPA treatment for ischemic stroke. Methods and Results: Male adult rats (330-380g) were subjected to embolic middle cerebral artery occlusion and then randomized into different treatment groups: saline injected at 4h; taurine (50 mg/kg) at 4h, tPA (10 mg/kg) at 6h, and combination therapy with taurine at 4h plus tPA at 6h after the onset of ischemia. Delayed 6-hour tPA did not decrease infarction but instead worsened brain hemorrhage. Combining taurine with delayed 6-hour tPA reduced tissue infarction, attenuated blood-brain barrier disruption by reduced degradation of tight junction proteins (claudin-5 and collagen IV), and ameliorated brain hemorrhage. These protective effects are correlated with decreased brain matrix metallopeptidase-9 activity possibly through inactivation of NF-kB and CD147 (a potent inducer of MMPs) in the postischemic brain, particularly in the cerebral microvessels (using structurally intact microvessels isolated from the brain). Conclusion: The present study demonstrates for the first time that combination therapy with taurine may attenuate delayed tPA-associated hemorrhagic transformation and extend tPA treatment time windows in a clinically relevant thromboembolic stroke model. Inhibition of MMP-9 in brain microvessels via inactivation of NF-κB and CD147 signaling may underlie the protective mechanisms of taurine.

Gap Junctions Suppress Electrical but Not [Ca2+] Heterogeneity in Resistance Arteries

Despite stochastic variation in the molecular composition and morphology of individual smooth muscle and endothelial cells, the membrane potential along intact microvessels is remarkably uniform. This is crucial for coordinated vasomotor responses. To investigate how this electrical homogeneity arises, a virtual arteriole was developed that introduces variation in the activities of ion-transport proteins between cells. By varying the level of heterogeneity and subpopulations of gap junctions (GJs), the resulting simulations shows that GJs suppress electrical variation but can only reduce cytosolic [Ca2+] variation. The process of electrical smoothing, however, introduces an energetic cost due to permanent currents, one which is proportional to the level of heterogeneity. This cost is particularly large when electrochemically different endothelial-cell and smooth-muscle-cell layers are coupled. Collectively, we show that homocellular GJs in a passively open state are crucial for electrical uniformity within the given cell layer, but homogenization may be limited by biophysical or energetic constraints. Owing to the ubiquitous presence of ion transport-proteins and cell-cell heterogeneity in biological tissues, these findings generalize across most biological fields.

Ischemic stroke‐induced increases in circulating microparticles promote leukocyte adhesion in intact microvessels (672.3)

Studies in both stroke patients and experimental animals indicate an association between ischemic stroke and post stroke peripheral inflammation, but the mechanisms are poorly understood. Our ongoing study indicated that circulating microparticles (MPs), the small vesicles released from the cell membrane upon activation, were significantly elevated in both patients and animals with diabetes, and increased circulating MPs with phosphatidylserine (PS) exposure on their outer membrane played important roles in the development of diabetes‐associated microvascular complications. The objective of this study was to investigate whether local ischemic stroke produced increased circulating MPs and determine their impact on post stroke‐associated peripheral vascular inflammation. Acute cerebral ischemia was induced by occlusion of the right middle cerebral artery (MCAO) in 3‐4 month old rats using an autologous blood clot. Functional assessment, lesion volume, and hemispheric swelling were evaluated at 24h after MCAO. Plasma MPs 24h after stroke induction were quantified and characterized using flow cytometry. The number of PS exposed MPs in stroke plasma, identified by Annexin V binding, significantly increased from 5.0 x 103 (normal rats) to 30 x 103 per µl. Antibodies directly against cell specific antigens were used to differentiate the MP cell origin. Results showed that 67% of the increased stroke plasma MPs were derived from platelets. Perfusion of individually cannulated normal rat mesenteric microvessels with isolated stroke MPs for 30 min followed by 10 min of resumed blood flow induced a 4‐fold increase in leukocyte adhesion compared to baseline. These results indicate that stroke‐induced increased circulating MPs serve as mediators capable of disseminating local inflammation to remote vasculatures, and therefore, promoting post stroke systemic complications.Grant Funding Source: Supported by HL56237, DK097391, NS061954

Advanced EUS Imaging for Pancreatic Diseases: CE-EUS (Low-MI, High-MI) and Real-Time Elastography, 3D

New advanced endosonographic techniques have only been introduced recently and will be demonstrated in this video sequence using the pancreatic organ as a target. The aim of these new techniques is to provide more information about a certain lesion in order to differentiate these lesions into benign and malignant. Although definitive differentiation is still a matter of histology, these new techniques are able to greatly increase the chance of correct diagnosis. Elastography is a technique which allows the estimation of the hardness of a lesion. The major problem of the technique is, however, that a hard structure is not necessarily malignant. Contrast-enhanced high mechanical index color Doppler endosonography was introduced shortly after the development of the ultrasound contrast enhancer. It provides the endosonographist vital information about the macrovessel system of the pancreas and can therefore discriminate inflammatory from malignant pancreatic lesions with a high degree of certainty. Contrast-enhanced low mechanical index endosonography is newly introduced into clinical practice and can display the vascularization of pancreatic microvessels. Because there are some cancerous lesions with an intact microvessel system, the degree of certainty is not as high as the high mechanical index version; however, it is still useful for clinical practice. The best results with the method can be achieved by using it for discrimination of cystic pancreatic lesions because of the excellent resolution of the method.Both methods can be used for a simultaneous three-dimensional (3D) reconstruction. The 3D reconstruction helps to mark the outlines of a lesion, and the data can be used to scroll through the lesion after the procedure is finished. This article is part of an expert video encyclopedia.