Synthetic microvascular networks for quantitative analysis of particle adhesion

We have developed a methodology to study particle adhesion in the microvascular environment using microfluidic, image-derived microvascular networks on a chip accompanied by Computational Fluid Dynamics (CFD) analysis of fluid flow and particle adhesion. Microfluidic networks, obtained from digitization of in vivo microvascular topology were prototyped using soft-lithography techniques to obtain semicircular cross sectional microvascular networks in polydimethylsiloxane (PDMS). Dye perfusion studies indicated the presence of well-perfused as well as stagnant regions in a given network. Furthermore, microparticle adhesion to antibody coated networks was found to be spatially non-uniform as well. These findings were broadly corroborated in the CFD analyses. Detailed information on shear rates and particle fluxes in the entire network, obtained from the CFD models, were used to show global adhesion trends to be qualitatively consistent with current knowledge obtained using flow chambers. However, in comparison with a flow chamber, this method represents and incorporates elements of size and complex morphology of the microvasculature. Particle adhesion was found to be significantly localized near the bifurcations in comparison with the straight sections over the entire network, an effect not observable with flow chambers. In addition, the microvascular network chips are resource effective by providing data on particle adhesion over physiologically relevant shear range from even a single experiment. The microfluidic microvascular networks developed in this study can be readily used to gain fundamental insights into the processes leading to particle adhesion in the microvasculature.     

Adhesion of bacteria to resected human colonic tissue: quantitative analysis of bacterial adhesion and viability

Adhesion to the intestinal mucosa is considered to be one of the main selection criteria of lactic acid bacteria for probiotic use. Adhesive probiotics are, for example, considered to provide better antagonism against pathogenic bacteria when compared to non-adhesive strains. Here a new model is described for studying adhesion and interaction of probiotic and pathogenic bacteria in the intestinal mucus in which the intestinal microbiota is present. The model is based on the use of human intestinal tissue, fluorescent-tagged bacteria and confocal laser scanning microscopy (CLSM) in adhesion measurements as well as human intestinal mucus and bioluminescent-tagged bacteria in viability measurements. Use of CLSM enabled, for the first time, real-time three-dimensional observations of live probiotic bacteria in their natural environment, the intestinal mucosa. When the real-time measurement of bacterial adhesion was combined with the real-time sensitive measurement of bacterial viability, it could be studied whether or not the adherent pathogens were alive. The model was used to study the interaction between Lactobacillus rhamnosus GG and Salmonella enterica serovar Typhimurium. We show that L. rhamnosus GG did not affect the adhesion or the viability of S. enterica serovar Typhimurium. Instead S. enterica serovar Typhimurium was shown to decrease the adhesion of L. rhamnosus GG in displacement assays. Moreover, the method is suitable for studies in which the interaction of two or more bacteria is examined in an environment in which other bacteria are present.     

Effect of platelet-activating factor on leukocyte adhesion to microvascular endothelium

The hamster cheek pouch microcirculation was used to investigate the effects of platelet-activating factor (PAF) on leukocyte adhesion to microvascular walls by means of intravital microscopy. PAF was applied topically at concentrations ranging from 10^–11 to 10^–5 M. An inverse relationship between PAF concentration and number of adhering white cells per 100-m length was found in venules ranging in diameter from 10 to 60 m (grouped into 10-m intervals). Importantly, the PAF-induced adhesion of leukocytes lasted for the 3-h experimental period. We postulate that induction of leukocyte adhesion to venular endothelium is an important role of PAF in inflammatory processes.     

Prognostic utility of quantitative image analysis of microvascular density in prostate cancer

The walls of angiogenic blood vessel capillaries are composed of two principal cell types, blood vessel endothelial cells (BEC) and pericytes (PC), whereas the walls of lymphatic capillaries are composed of lymphatic endothelial cells (LEC). In this investigation we describe a practical application of NIH ImageJ software for quantitative image analysis for pericytes and endothelial cells in prostate cancer. We used a tissue microarray that contained 49 tissue cores (normal prostate tissue or prostatic carcinomas with Gleason scores of 6 through 10). These prostate cancer samples represented AJCC prognostic stages II, III, and IV. Slides were immunostained with anti‐PDGFR‐β antibody for identification of PC, and quantified as microvascular pericyte density (MVPD); they were also immunostained with anti‐CD34 antibody for identification of LEC and BEC simultaneously, and quantified as microvascular endothelial density (MVED). CD31 and D2‐40 immunostains were used to quantify BEC and lymphatic endothelial cells, respectively. Our results showed higher MVPD and MVED in prostate cancers with higher Gleason scores and higher stages, suggesting the prognostic utility of vascular image analysis in prostate pathology. This investigation demonstrates the feasibility, versatility, and ease of use of ImageJ software and pericyte‐specific and endothelial‐specific immunohistochemistry for quantitative image analysis in prostate pathology.     

Microfabrication of cylindrical microfluidic channel networks for microvascular research

Current methods for formation of microvascular channel scaffolds are limited with non-circular channel cross-sections, complicated fabrication, and less flexibility in microchannel network design. To address current limitations in the creation of engineered microvascular channels with complex three-dimensional (3-D) geometries in the shape of microvessels, we have developed a reproducible, cost-effective, and flexible micromanufacturing process combined with photolithographic reflowable photoresist and soft lithography techniques to fabricate cylindrical microchannel and networks. A positive reflowable photoresist AZ P4620 was used to fabricate a master microchannel mold with semi-circular cross-sections. By the alignment and bonding of two polydimethylsiloxane (PDMS) microchannels replicated from the master mold together, a cylindrical microchannel or microchannel network was created. Further examination of the channel dimensions and surface profiles at different branching levels showed that the shape of the microfluidic channel was well approximated by a semi-circular surface, and a multi-level, multi-depth channel network was created. In addition, a computational fluidic dynamics (CFD) model was used to simulate shear flows and corresponding pressure distributions inside of the microchannel and channel network based on the dimensions of the fabricated channels. The fabricated multi-depth cylindrical microchannel network can provide platforms for the investigation of microvascular cells growing inside of cylindrical channels under shear flows and lumen pressures, and work as scaffolds for the investigation of morphogenesis and tubulogenesis.     

Human microvascular lymphatic and blood endothelial cells produce fibrillin: deposition patterns and quantitative analysis

Fibrillin microfibrils constitute a scaffold for elastin deposition in the wall of arteries and form the anchoring filaments that connect the lymphatic endothelium to surrounding elastic fibers. We previously reported that fibrillin is deposited in a honeycomb pattern in bovine arterial endothelial cells, which also deposit microfibril-associated glycoprotein (MAGP)-1, whereas thoracic duct endothelial cells form an irregular web. The present immunohistochemical study was designed to verify whether lymphatic and blood human dermal microvascular endothelial cells (HDMECs) isolated from human foreskin by the sequential use of a pan-endothelial marker, CD31, and the lymphatic specific marker, D2-40, deposit fibrillin and MAGP-1. In both cell types, fibrillin and MAGP-1 co-localized and were deposited with different patterns of increasing complexity co-existing in the same culture. Fibrillin microfibrils formed a wide-mesh honeycomb leaving fibrillin-free spaces that were gradually filled. This modality of fibrillin deposition, similar to that of bovine large artery endothelial cells, was basically the same in blood and lymphatic HDMECs. In some lymphatic HDMECs, fibrillin was initially deposited as uniformly scattered short fibrillin strands probably as a result of anchoring filaments carried over from the vessels of origin. Our findings show that blood and lymphatic endothelial cells participate in fibrillin deposition in human skin.     

A two-fluid model for hematocrit distribution in microvascular networks

A new theoretical network model for evaluating discharge hematocrits, explicitly based on plasma skimming at branches, is introduced. The particular network geometry chosen simulates bat wing microvasculature. Blood in vessels is approximated to be a two fluid with red cell suspension as the core and a plasma layer surrounding it. The plasma layer width depends on hematocrit, which leads to nonlinear hydrodynamic equations solved by iteration. Discharge hematocrit distributions are calculated by a computer for five generations of vessels. Dispersion of hematocrit values was found to be correlated to plasma skimming at branches. Contrary to previous suggestions, plasma skimming did not result in lowered mean hematocrit towards the capillaries. Network structure was found to be an important factor affecting the hematocrits. Mean discharge hematocrit remained steady against changes in vessel dimensions, capillary resistances, red cell concentration in plasma layer, and shape of the separation surface defining the streamlines entering the side branch. This high stable mean hematocrit is based on symmetry of the model network. Enhanced asymmetry tended to lower the hematocrit.     

Informational dynamics of vasomotion in microvascular networks: a review

Vasomotion refers to spontaneous oscillation of small vessels observed in many microvascular beds. It is an intrinsic phenomenon unrelated to cardiac rhythm or neural and hormonal regulation. Vasomotion is found to be particularly prominent under conditions of metabolic stress. In spite of a significant existent literature on vasomotion, its physiological and pathophysiological roles are not clear. It is thought that modulation of vasomotion by vasoactive substances released by metabolizing tissue plays a role in ensuring optimal delivery of nutrients to the tissue. Vasomotion rhythms exhibit a great variety of temporal patterns from regular oscillations to chaos. The nature of vasomotion rhythm is believed to be significant to its function, with chaotic vasomotion offering several physiological advantages over regular, periodic vasomotion. In this article, we emphasize that vasomotion is best understood as a network phenomenon. When there is a local metabolic demand in tissue, an ideal vascular response should extend beyond local microvasculature, with coordinated changes over multiple vascular segments. Mechanisms of information transfer over a vessel network have been discussed in the literature. The microvascular system may be regarded as a network of dynamic elements, interacting, either over the vascular anatomical network via gap junctions, or physiologically by exchange of vasoactive substances. Drawing analogies with spatiotemporal patterns in neuronal networks of central nervous system, we ask if properties like synchronization/desynchronization of vasomotors have special significance to microcirculation. Thus the contemporary literature throws up a novel view of microcirculation as a network that exhibits complex, spatiotemporal and informational dynamics.     

A quantitative analysis of the microvascular sequestration of malaria parasites in the human brain.

Microvascular sequestration was assessed in the brains of 50 Thai and Vietnamese patients who died from severe malaria (Plasmodium falciparum, 49; P. vivax, 1). Malaria parasites were sequestered in 46 cases; in 3 intravascular malaria pigment but no parasites were evident; and in the P. vivax case there was no sequestration. Cerebrovascular endothelial expression of the putative cytoadherence receptors ICAM-1, VCAM-1, E-selectin, and chondroitin sulfate and also HLA class II was increased. The median (range) ratio of cerebral to peripheral blood parasitemia was 40 (1.8 to 1500). Within the same brain different vessels had discrete but different populations of parasites, indicating that the adhesion characteristics of cerebrovascular endothelium change asynchronously during malaria and also that significant recirculation of parasitized erythrocytes following sequestration is unlikely. The median (range) ratio of schizonts to trophozoites (0.15:1; 0.0 to 11.7) was significantly lower than predicted from the parasite life cycle (P < 0.001). Antimalarial treatment arrests development at the trophozoite stages which remain sequestered in the brain. There were significantly more ring form parasites (age < 26 hours) in the cerebral microvasculature (median range: 19%; 0-90%) than expected from free mixing of these cells in the systemic circulation (median range ring parasitemia: 1.8%; 0-36.2%). All developmental stages of P. falciparum are sequestered in the brain in severe malaria.     

Effect of ultraviolet light on the expression of adhesion molecules and T lymphocyte adhesion to human dermal microvascular endothelial cells

In order to determine the effect of ultraviolet radiation (UVR) on the cell adhesion molecules expressed in human dermal microvascular endothelial cells (HDMEC), the cells were exposed to varying UVR doses and the cell surface was examined for expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM- 1), and E-selectin. The effect of UVB irradiation on the binding of T lymphocytes to HDMEC was also examined. UVA irradiation did not affect the surface expression of ICAM-1, VCAM-1, or E-selectin on the HDMEC. However, following UVB exposure, ELISA demonstrated a significant increase in the baseline ICAM-1 cell surface expression on the HDMEC. However, no induction of either E-selectin or VCAM-1 was noted. UVB also significantly augmented ICAM-1 induction by IL-1alpha and TNF-alpha. VCAM-1 was induced by stimulating HDMEC with IL-1alpha following a UVB irradiation dose of 100 mJ/cm2. Flow cytometric analysis of the HDMEC stimulated with IL-1alpha for 24h demonstrated that 12% of the cells expressed VCAM-1 but either IL-1alpha or UVB irradiation alone failed to induce VCAM-1 expression. Enhancement of T cell-HDMEC binding by IL-1alpha or TNF-alpha treatment was not significantly affected after UVB irradiation. This study demonstrated that UVB irradiation can alter ICAM-1 and VCAM-1 expression on the HDMEC surface and that augmentation of ICAM-1 expression and the IL-1alpha-dependent induction of VCAM-1 following UVB exposure might be important steps in the pathogenesis of sunburn.     

RBF networks for source localization in quantitative electrophysiology

The backpropagation neural network methods have been proposed recently to solve the inverse problem in quantitative electrophysiology. A major advantage of the technique is that once a neural network is trained, it no longer requires iterations or access to sophisticated computations. We propose to use RBF networks for source localization in the brain, and systematically compare their performance to those of Levenberg-Marquardt (LM) algorithms. We show the use of two types of Radial Basis Function Networks (RBF) network: a classic network with fixed number of hidden layer neurons and an improved network, Minimal Resource Allocation Network (MRAN), recently proposed by one of the authors, capable for dynamically configuring its structure so as to obtain a compact topology to match the data presented to it.     

Thromboxane-induced neutrophil adhesion to pulmonary microvascular and aortic endothelium is regulated by CD18

Thromboxane (Tx) A₂ generation and subsequent selective pulmonary sequestration of neutrophils (PMNs) is characteristic of several forms of the adult respiratory distress syndrome (ARDS). Therefore, we examined PMN-dependent adhesion to cultured pulmonary microvessel and aortic endothelium (EC) in response to U46,619 (Tx mimic). Nonstimulated PMNs were two fold more adherent to pulmonary microvessel HC than to aortic EC (P < 0.01). PMN pretreatment with Tx mimic (10^–6 M) increased adhesion to both types of EC (P < 0.01). The Tx mimic-induced adhesion was blocked by receptor antagonists to Tx (SQ29,548) and to leukotrienes (FPL55,712), and by the anti-CD18 mAb TS1/18 (P < 0.01, all cases). Baseline PMN adhesion also was modulated by Tx, leukotrienes, and CD18, for both EC types. These results indicate pulmonary microvessel EC is intrinsically more adhesive for both nonstimulated and stimulated PMNs than aortic EC and that Tx mediates PMN-dependent adhesion by coupled interaction of Tx and LT receptors via CD 18 activation.This research was supported in part by: USPHS, NHLBI 16714, 43875, 33104, and GM24891.     

A physiologically realistic in vitro model of microvascular networks

Existing microfluidic devices, e.g. parallel plate flow chambers, do not accurately depict the geometry of microvascular networks in vivo. We have developed a synthetic microvascular network (SMN) on a polydimethalsiloxane (PDMS) chip that can serve as an in vitro model of the bifurcations, tortuosities, and cross-sectional changes found in microvascular networks in vivo. Microvascular networks from a cremaster muscle were mapped using a modified Geographical Information System, and then used to manufacture the SMNs on a PDMS chip. The networks were cultured with bovine aortic endothelial cells (BAEC), which reached confluency 3–4 days after seeding. Propidium iodide staining indicated viable and healthy cells showing normal behavior in these networks. Anti-ICAM-1 conjugated 2-μm microspheres adhered to BAEC cells activated with TNF-α in significantly larger numbers compared to control IgG conjugated microspheres. This preferential adhesion suggests that cultured cells retain an intact cytokine response in the SMN. This microfluidic system can provide novel insight into characterization of drug delivery particles and dynamic flow conditions in microvascular networks.