Fluid and protein exchange in microvascular networks: Importance of modelling heterogeneity in geometrical and biophysical properties.

KEY POINTS: Microvascular network architecture defines coupling of fluid and protein exchange. Network arrangements markedly reduce capillary hydrostatic pressures and resting fluid movement at the same time as increasing the capacity for change The presence of vascular remodelling or angiogenesis puts constraints of network behaviour The sites of fluid and protein exchange can be segregated to different portions of the network Although there is a net filtration of fluid from a network of exchange vessels, there are specific areas where fluid moves into the circulation (reabsorption) and, when protein is moving into tissue, the amount is insufficient under basal conditions to result in changes in oncotic pressure. ABSTRACT: Integration of functional results obtained across scales, from chemical signalling to the whole organism, is a daunting task requiring the marriage of experimental data with mathematical modelling. In the present study, a novel coupled computational fluid dynamics model is developed incorporating fluid and protein transport using measurements in an in vivo frog (Rana pipiens) mesenteric microvascular network. The influences of network architecture and exchange are explored systematically under the common assumptions of structurally and functionally identical microvessels (Homogeneous Scenario) or microvessels classified by position in flow (Class Uniform Scenario), which are compared with realistic microvascular network components (Heterogeneous Scenario). The model incorporates ten quantities that vary within a microvessel; pressure boundary conditions are calibrated against experimental measurements. The Homogeneous Scenario standard model showed that assuming a single 'typical' capillary hides the influence of vessels arranged into a network architecture, where capillary hydrostatic pressures (pT ) are reduced, resulting in both a nonuniform distribution of blood flow and reduced volume flow rate (Jf,T ). In the Class Uniform Scenario pT was further attenuated to produce a ∼60% reduction in Jf,T . Finally, the Heterogeneous Scenario, incorporating measures of individual vessel surface area, demonstrates additional lowering of pT from inlet values favouring a >70% reduction of Jf,T in the face of a ∼120% increase in protein movement into the tissues relative to the Homogeneous Scenario. Beyond the impacts of network architecture, an unanticipated finding was the influence of a blind-end microvessel on model convergence, indicating a profound influence of the largely unexplored dynamics of vascular remodelling on tissue perfusion.

Sex differences influencing micro- and macrovascular endothelial phenotype in vitro.

KEY POINTS: Endothelial dysfunction is an early hallmark of multiple disease states that also display sex differences with respect to age of onset, frequency and severity. Results of in vivo studies of basal and stimulated microvascular barrier function revealed sex differences that are difficult to ascribe to specific cells or environmental factors. The present study evaluated endothelial cells (EC) isolated from macro- and/or microvessels of reproductively mature rats under the controlled conditions of low-passage culture aiming to test the assumption that EC phenotype would be sex independent. The primary finding was that EC, regardless of where they are derived, retain a sex-bias in low-passage culture, independent of varying levels of reproductive hormones. The implications of the present study include the fallacy of expecting a universal set of mechanisms derived from study of EC from one sex and/or one vascular origin to apply uniformly to all EC under unstimulated conditions, and no less in disease. ABSTRACT: Vascular endothelial cells (EC) are heterogeneous with respect to phenotype, reflecting at least the organ of origin, location within the vascular network and physical forces. As an independent influence on EC functions in health or aetiology, susceptibility, and progression of dysfunction in numerous disease states, sex has been largely ignored. The present study focussed on EC isolated from aorta (macrovascular) and skeletal muscle vessels (microvascular) of age-matched male and female rats under identical conditions of short-term (passage 4) culture. We tested the hypothesis that genomic sex would not influence endothelial growth, wound healing, morphology, lactate production, or messenger RNA and protein expression of key proteins (sex hormone receptors for androgen and oestrogens α and ß; platelet endothelial cell adhesion molecule-1 and vascular endothelial cadherin mediating barrier function; αv ß3 and N-cadherin influencing matrix interactions; intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 mediating EC/white cell adhesion). The hypothesis was rejected because the EC origin (macro- vs. microvessel) and sex influenced multiple phenotypic characteristics. Statistical model analysis of EC growth demonstrated an hierarchy of variable importance, recapitulated for other phenotypic characteristics, with predictions assuming EC homogeneity < sex < vessel origin < sex and vessel origin. Furthermore, patterns of EC mRNA expression by vessel origin and by sex did not predict protein expression. Overall, the present study demonstrated that accurate assessment of sex-linked EC dysfunction first requires an understanding of EC function by position in the vascular tree and by sex. The results from a single EC tissue source/species/sex cannot provide universal insight into the mechanisms regulating in vivo endothelial function in health, and no less in disease.

Sex-Specific Characteristics of the Microcirculation.

The requirements of metabolizing tissue are both continuous and variable; accordingly, the microvasculature serving that tissue must be similarly dynamic. Just as it is recognized that males and females of the same species have differing metabolic requirements, is it not likely that the microvasculature serving these tissues will differ by sex? This section focusing on the constituents of the microcirculation identifies what is known presently about the role sex plays in matching metabolic demand with microvascular function and areas requiring additional study. Many of the identified sex differences are subtle and easily ignored. In the aggregate, though, they can profoundly alter phenotype, especially under stressful conditions including pregnancy, exercise, and disease states ranging from diabetes to heart failure. Although the features presently identified to "have sex" range from differences in growth, morphology, protein expression, and intracellular signaling, males and females alike achieve homeostasis, likely by different means. Studies of microvascular sexual dimorphism are also identifying age as an independent but interacting factor requiring additional attention. Overall, attempting to ignore either sex and/or age is inappropriate and will prevent the design and implementation of appropriate interventions to present, ameliorate, or correct microvascular dysfunction.

Permeability and contractile responses of collecting lymphatic vessels elicited by atrial and brain natriuretic peptides.

Atrial and brain natriuretic peptides (ANP and BNP, respectively) are cardiac hormones released into the bloodstream in response to hypervolaemia or fluid shifts to the central circulation. The actions of both peptides include natriuresis and diuresis, a decrease in systemic blood pressure, and inhibition of the renin-angiotensin-aldosterone system. Further, ANP and BNP elicit increases in blood microvessel permeability sufficient to cause protein and fluid extravasation into the interstitium to reduce the vascular volume. Given the importance of the lymphatic vasculature in maintaining fluid balance, we tested the hypothesis that ANP or BNP (100 nM) would likewise elevate lymphatic permeability (Ps) to serum albumin. Using a microfluorometric technique adapted to in vivo lymphatic vessels, we determined that rat mesenteric collecting lymphatic Ps to rat serum albumin increased by 2.0 ± 0.4-fold (P = 0.01, n = 7) and 2.7 ± 0.8-fold (P = 0.07, n = 7) with ANP and BNP, respectively. In addition to measuring Ps responses, we observed changes in spontaneous contraction amplitude and frequency from the albumin flux tracings in vivo. Notably, ANP abolished spontaneous contraction amplitude (P = 0.005) and frequency (P = 0.006), while BNP augmented both parameters by ∼2-fold (P < 0.01 each). These effects of ANP and BNP on contractile function were examined further by using an in vitro assay. In aggregate, these data support the theory that an increase in collecting lymphatic permeability opposes the absorptive function of the lymphatic capillaries, and aids in the retention of protein and fluid in the interstitial space to counteract volume expansion.

Endothelial barrier dysfunction in diabetic conduit arteries: a novel method to quantify filtration.

The endothelial barrier plays an important role in atherosclerosis, hyperglycemia, and hypercholesterolemia. In the present study, an accurate, reproducible, and user-friendly method was used to further understand endothelial barrier function of conduit arteries. An isovolumic method was used to measure the hydraulic conductivity (L(p)) of the intact vessel wall and medial-adventitial layer. Normal arterial segments with diameters from 0.2 to 5.5 mm were used to validate the method, and femoral arteries of diabetic rats were studied as an example of pathological specimens. Various arterial segments confirmed that the volume flux of water per unit surface area was linearly related to intraluminal pressure, as confirmed in microvessels. L(p) of the intact wall varied from 3.5 to 22.1 × 10(-7) cm·s(-1)·cmH(2)O(-1) over the pressure range of 7-180 mmHg. Over the same pressure range, L(p) of the endothelial barrier changed from 4.4 to 25.1 × 10(-7) cm·s(-1)·cmH(2)O(-1). During perfusion with albumin-free solution, L(p) of rat femoral arteries increased from 6.1 to 13.2 × 10(-7) cm·s(-1)·cmH(2)O(-1) over the pressure range of 10-180 mmHg. Hyperglycemia increased L(p) of the femoral artery in diabetic rats from 2.9 to 5.5 × 10(-7) cm·s(-1)·cmH(2)O(-1) over the pressure range of 20-135 mmHg. In conclusion, the L(p) of a conduit artery can be accurately and reproducibly measured using a novel isovolumic method, which in diabetic rats is hyperpermeable. This is likely due to disruption of the endothelial glycocalyx.

Cardiovascular sex-differences: insights via physiology-based modeling and potential for noninvasive sensing via ballistocardiography.

In this study, anatomical and functional differences between men and women in their cardiovascular systems and how these differences manifest in blood circulation are theoretically and experimentally investigated. A validated mathematical model of the cardiovascular system is used as a virtual laboratory to simulate and compare multiple scenarios where parameters associated with sex differences are varied. Cardiovascular model parameters related with women's faster heart rate, stronger ventricular contractility, and smaller blood vessels are used as inputs to quantify the impact (i) on the distribution of blood volume through the cardiovascular system, (ii) on the cardiovascular indexes describing the coupling between ventricles and arteries, and (iii) on the ballistocardiogram (BCG) signal. The model-predicted outputs are found to be consistent with published clinical data. Model simulations suggest that the balance between the contractile function of the left ventricle and the load opposed by the arterial circulation attains similar levels in females and males, but is achieved through different combinations of factors. Additionally, we examine the potential of using the BCG waveform, which is directly related to cardiovascular volumes, as a noninvasive method for monitoring cardiovascular function. Our findings provide valuable insights into the underlying mechanisms of cardiovascular sex differences and may help facilitate the development of effective noninvasive cardiovascular monitoring methods for early diagnosis and prevention of cardiovascular disease in both women and men.

Lymphatic fluid: exchange mechanisms and regulation.

Regulation of fluid and material movement between the vascular space of microvessels penetrating functioning organs and the cells therein has been studied extensively. Unanswered questions as to the regulatory mechanisms and routes remain. Significantly less is known about the lymphatic vascular system given the difficulties in seeing, no less isolating, these vessels lying deeper in these same tissues. It has become evident that the exchange microvasculature is not simply a passive biophysical barrier separating the vascular and interstitial compartments but a dynamic, multicellular structure subject to acute regulation and chronic adaptation to stimuli including inflammation, sepsis, diabetes, injury, hypoxia and exercise. Similarly lymphatic vessels range, in their simplest form, from lymphatic endothelium attached to the interstitial matrix, to endothelia and phasic lymphatic smooth muscle that act as Starling resistors. Recent work has demonstrated that among the microvascular lymphatic elements, the collecting lymphatics have barrier properties similar to venules, and thus participate in exchange. As with venules, vasoactive agents can alter both the permeability and contractile properties thereby setting up previously unanticipated gradients in the tissue space and providing potential targets for the pharmacological prevention and/or resolution of oedema.

Reverse engineering of oxygen transport in the lung: adaptation to changing demands and resources through space-filling networks.

The space-filling fractal network in the human lung creates a remarkable distribution system for gas exchange. Landmark studies have illuminated how the fractal network guarantees minimum energy dissipation, slows air down with minimum hardware, maximizes the gas- exchange surface area, and creates respiratory flexibility between rest and exercise. In this paper, we investigate how the fractal architecture affects oxygen transport and exchange under varying physiological conditions, with respect to performance metrics not previously studied.We present a renormalization treatment of the diffusion-reaction equation which describes how oxygen concentrations drop in the airways as oxygen crosses the alveolar membrane system. The treatment predicts oxygen currents across the lung at different levels of exercise which agree with measured values within a few percent. The results exhibit wide-ranging adaptation to changing process parameters, including maximum oxygen uptake rate at minimum alveolar membrane permeability, the ability to rapidly switch from a low oxygen uptake rate at rest to high rates at exercise, and the ability to maintain a constant oxygen uptake rate in the event of a change in permeability or surface area. We show that alternative, less than space-filling architectures perform sub-optimally and that optimal performance of the space-filling architecture results from a competition between underexploration and overexploration of the surface by oxygen molecules.

Microvascular Sex- and Age- Dependent Phosphodiesterase Expression.

Objective: The cyclic nucleotide second messengers, cAMP and cGMP, are pivotal regulators of vascular functions; their cellular levels are tightly controlled by the cyclic nucleotide hydrolases, phosphodiesterases (PDE). Biologic sex and age are recognized as independent factors impacting the mechanisms mediating both vascular health and dysfunction. This study focused on microvessels isolated from male and female rats before (juvenile) and after (adult) sexual maturity under resting conditions. We tested the hypothesis that sexual dimorphism in microvascular PDE expression would be absent in juvenile rats, but would manifest in adult rats. Methods: Abdominal skeletal muscle arterioles and venules were isolated from age-matched juvenile and adult male and female rats under resting conditions. Transcripts of five PDE families (1-5) associated with coronary and vascular function with a total of ten genes were measured using TaqMan real-time RT-PCR and protein expression of microvessel PDE4 was assessed using immunoblotting and immunofluorescence. Results: Overall expression levels of PDE5A were highest while PDE3 levels were lowest among the five PDE families (p < 0.05) regardless of age or sex. Contrary to our hypothesis, in juveniles, sexual dimorphism in PDE expression was observed in three genes: arterioles (PDE1A, female > male) and venules (PDE1B and 3A, male > female). In adults, gene expression levels in males were higher than females for five genes in arterioles (PDE1C, 3A, 3B, 4B, 5A) and three genes (PDE3A, 3B, and 5A) in venules. Furthermore, age-related differences were observed in PDE1-5 (in males, adult > juvenile for most genes in arterioles; in females, adult > juvenile for arteriolar PDE3A; juvenile gene expression > adult for two genes in arterioles and three genes in venules). Immunoblotting and immunofluorescence analysis revealed protein expression of microvessel PDE4. Conclusion: This study revealed sexual dimorphism in both juvenile and adult rats, which is inconsistent with our hypothesis. The sex- and age-dependent differences in PDE expression implicate different modulations of cAMP and cGMP pathways for microvessels in health. The implication of these sex- and age-dependent differences, as well as the duration and microdomain of PDE1-5 activities in skeletal muscle microvessels, in both health and disease, require further investigation.

Multi-focus Image Fusion for Confocal Microscopy Using U-Net Regression Map.

Characterizing the spatial relationship between blood vessel and lymphatic vascular structures, in the mice dura mater tissue, is useful for modeling fluid flows and changes in dynamics in various disease processes. We propose a new deep learning-based approach to fuse a set of multi-channel single-focus microscopy images within each volumetric z-stack into a single fused image that accurately captures as much of the vascular structures as possible. The red spectral channel captures small blood vessels and the green fluorescence channel images lymphatics structures in the intact dura mater attached to bone. The deep architecture Multi-Channel Fusion U-Net (MCFU-Net) combines multi-slice regression likelihood maps of thin linear structures using max pooling for each channel independently to estimate a slice-based focus selection map. We compare MCFU-Net with a widely used derivative-based multi-scale Hessian fusion method [8]. The multi-scale Hessian-based fusion produces dark-halos, non-homogeneous backgrounds and less detailed anatomical structures. Perception based no-reference image quality assessment metrics PIQUE, NIQE, and BRISQUE confirm the effectiveness of the proposed method.

In vivo determination of collecting lymphatic vessel permeability to albumin: a role for lymphatics in exchange.

While it is well established that the lymphatic vasculature is central to fluid and solute homeostasis, how it accomplishes this task is not well defined. To clarify the basic mechanisms underlying basal fluid and solute homeostasis, we assessed permeability to rat serum albumin (P(RSA)(s)) in mesenteric collecting lymphatic vessels and venules of juvenile male rats. Using the quantitative microfluorometric technique originally developed for blood capillaries, we tested the hypothesis that as a consequence of venules and collecting lymphatics sharing a common embryological origin, their P(RSA)(s) would not differ significantly. Supporting our hypothesis, the median collecting lymphatic P(RSA)(s) (3.5 +/- 1.0 x 10(7) cm s(-1), N = 22) did not differ significantly from the median venular P(RSA)(s) (4.0 +/- 1.0 x 10(7) cm s(-1), N = 8, P = 0.61). For collecting lymphatics the diffusive permeability (P(d) = 2.5 x 10(7) cm s(-1)) was obtained from the relationship of apparent P(RSA)(s) and pressure. While the measured P(RSA)(s), P(d) and estimated hydraulic conductivity of collecting lymphatics and venules were similar, the contribution of convective coupling differs as a result of the higher hydrostatic pressure experienced by venules relative to collecting lymphatics in vivo. In summary, the data demonstrate the capacity for collecting lymphatics to act as exchange vessels, able to extravasate solute and filter fluid. As a consequence these data provide experimental support for the theory that prenodal lymphatic vessels concentrate intraluminal protein.

Intrinsic sex-specific differences in microvascular endothelial cell phosphodiesterases.

The importance of gonadal hormones in the regulation of vascular function has been documented. An alternate and essential contribution of the sex chromosomes to sex differences in vascular function is poorly understood. We reported previously sex differences in microvessel permeability (P(s)) responses to adenosine that were mediated by the cAMP signaling pathway (Wang J, PhD thesis, 2005; Wang J and Huxley V, Proceedings of the VIII World Congress of Microcirculation, 2007; Wang J and Huxley VH, Am J Physiol Heart Circ Physiol 291: H3094-H3105, 2006). The two cyclic nucleotides, cAMP and cGMP, central to the regulation of vascular barrier integrity, are hydrolyzed by phosphodiesterases (PDE). We hypothesized that microvascular endothelial cells (EC) would retain intrinsic and inheritable sexually dimorphic genes with respect to the PDEs modulating EC barrier function. Primary cultured microvascular EC from skeletal muscles isolated from male and female rats, respectively, were used. SRY (a sex-determining region Y gene) mRNA expression was observed exclusively in male, not female, cells. The predominant isoform among PDE1-5, present in both XY and XX EC, was PDE4. Expression mRNA levels of PDE1A (male > female) and PDE3B (male < female) were sex dependent; PDE2A, PDE4D, and PDE5A were sex independent. Barrier function, P(s), was determined from measures of albumin flux across confluent primary cultured microvessel XY and XX EC monolayers. Consistent with intact in situ microvessels, basal monolayer P(s) did not differ between XY (1.7 +/- 0.2 x 10(-6) cm/s; n = 8) and XX (1.8 +/- 0.1 x 10(-6) cm/s; n = 10) EC. Cilostazol, a PDE3 inhibitor, reduced (11%, P < 0.05) P(s) in XX, not XY, cells. These findings demonstrate the presence and maintenance of intrinsic sex-related differences in gene expression and cellular phenotype by microvascular EC in a gonadal-hormone-free environment. Furthermore, intrinsic cell-sex likely contributes significantly to sexual dimorphism in cardiovascular function.

Corrections to "Cardiovascular Function and Ballistocardiogram: A Relationship Interpreted via Mathematical Modeling" [Oct 19 2906-2917].

Presents a missing funding acknowledgment for Dr. Mihail Popescu in the above named paper.

Complex Non-sinus-associated Pachymeningeal Lymphatic Structures: Interrelationship With Blood Microvasculature.

The contribution of cranial dura mater vascular networks, as means for maintaining brain fluid movement and balance, and as the source of significant initiators and/or contributors to neurological disorders, has been overlooked. These networks consist of both blood and lymphatic vessels. The latter were discovered recently and described as sinus-associated structures thus changing the old paradigm that central nervous system lacks lymphatics. In this study, using markers specific to blood and lymphatic endothelia, we demonstrate the existence of the complex non-sinus-associated pachymeningeal lymphatic vasculature. We further show the interrelationship and possible connections between lymphatic vessels and the dural blood circulatory system. Our novel findings reveal the presence of lymphatic-like structures that exist on their own and/or in close proximity to microvessels. Of particular interest are sub-sets of vascular complexes with dual (lymphatic and blood) vessel identity representing a unique microenvironment within the cranial dura. The close association of the systemic blood circulation and meningeal lymphatics achieved in these complexes could facilitate fluid exchange between the two compartments and constitute an alternative route for CSF drainage.

Cardiovascular Function and Ballistocardiogram: A Relationship Interpreted via Mathematical Modeling.

OBJECTIVE: To develop quantitative methods for the clinical interpretation of the ballistocardiogram (BCG). METHODS: A closed-loop mathematical model of the cardiovascular system is proposed to theoretically simulate the mechanisms generating the BCG signal, which is then compared with the signal acquired via accelerometry on a suspended bed. RESULTS: Simulated arterial pressure waveforms and ventricular functions are in good qualitative and quantitative agreement with those reported in the clinical literature. Simulated BCG signals exhibit the typical I, J, K, L, M, and N peaks and show good qualitative and quantitative agreement with experimental measurements. Simulated BCG signals associated with reduced contractility and increased stiffness of the left ventricle exhibit different changes that are characteristic of the specific pathological condition. CONCLUSION: The proposed closed-loop model captures the predominant features of BCG signals and can predict pathological changes on the basis of fundamental mechanisms in cardiovascular physiology. SIGNIFICANCE: This paper provides a quantitative framework for the clinical interpretation of BCG signals and the optimization of BCG sensing devices. The present paper considers an average human body and can potentially be extended to include variability among individuals.

Patch-Based Semantic Segmentation for Detecting Arterioles and Venules in Epifluorescence Imagery.

Segmentation and quantification of microvasculature structures are the main steps toward studying microvasculature remodeling. The proposed patch based semantic architecture enables accurate segmentation for the challenging epifluorescence microscopy images. Our pixel-based fast semantic network trained on random patches from different epifluorescence images to learn how to discriminate between vessels versus nonvessels pixels. The proposed semantic vessel network (SVNet) relies on understanding the morphological structure of the thin vessels in the patches rather than considering the whole image as input to speed up the training process and to maintain the clarity of thin structures. Experimental results on our ovariectomized - ovary removed (OVX) - mice dura mater epifluorescence microscopy images shows promising results in both arteriole and venule part. We compared our results with different segmentation methods such as local, global thresholding, matched based filter approaches and related state of the art deep learning networks. Our overall accuracy (> 98%) outperforms all the methods including our previous work (VNet). [1].

Sex and the cardiovascular system: the intriguing tale of how women and men regulate cardiovascular function differently.

The ability to recognize and appreciate from a reproductive standpoint that males and females possess different attributes has been long standing. Only more recently have we begun to look more deeply into both the similarities and differences between men and women, as well as between boys and girls, with respect to the structure and function of other organ systems. This article focuses on the cardiovascular system, with examples of sex differences in the control of coronary function, blood pressure, and volume. Recognizing the differences between the sexes with respect to cardiovascular function facilitates understanding of the mechanisms whereby homeostasis can be achieved using different contributions or components of the living system. Furthermore, recognition of the differences as well as the similarities permits the design of appropriate diagnostic instruments, recognition of sex-specific pathophysiology, and implementation of appropriate treatment of cardiovascular disease in men and women.

Estrogen-Dependent Changes in Dura Mater Microvasculature Add New Insights to the Pathogenesis of Headache.

The pathogenesis of headaches is a matter of ongoing discussion of two major theories describing it either as a vascular phenomenon resulting from vasodilation or primarily as a neurogenic process accompanied by secondary vasodilation associated with sterile neurogenic inflammation. While summarizing current views on neurogenic and vascular origins of headache, this mini review adds new insights regarding how smooth muscle-free microvascular networks, discovered within dura mater connective tissue stroma (previously thought to be "avascular"), may become a site of initial insult generating the background for the development of headache. Deficiencies in estrogen-dependent control of microvascular integrity leading to plasma protein extravasation, potential activation of perivascular and connective tissue stroma nociceptive neurons, and triggering of inflammatory responses are described. Finally, possible avenues for controlling and preventing these pathophysiological changes are discussed.

Permeability response of the rat mesenteric microvasculature to insulin.

Whether insulin influences microvascular exchange is important in understanding its specific role in insulin resistance and the treatment of diabetes. We investigated whether insulin could induce changes in the microvascular flux of albumin from the mesenteric venules of anesthetized male and female Sprague-Dawley rats (n = 11). After catheterization for monitoring of mean arterial pressure (MAP), a loop of small intestine was exteriorized. The mesentery was draped over a coverslip for observation and suffused continuously with bicarbonate-buffered solution (BBS) (pH 7.4, 37 degrees C). After intravenous injection of Alexa 594trade mark labeled bovine serum albumin (BSA, 8 mg/kg), fluorescence intensity (I(f)) was recorded on videotape for 30 minutes BBS suffusion and 75 minutes suffusion of BBS plus 0.02 U/ml porcine insulin. Microvascular flux of BSA was measured as a leak index (LI) of I(f) in a 10 x 30 mum window over a postcapillary venule relative to I(f) of the adjoining tissue. Insulin induced a rapid 34% decrease in LI within 5 minutes (p<0.05) that was sustained for the next 30 minutes. We also observed gender and age differences in the permeability response to insulin, as there was a sustained approximately 59% decrease in LI in adult females (n = 5) after 25 minutes, whereas there was an acute, transient (15 min) 45% decrease in LI in juvenile males (n = 6). We conclude that insulin reduces mesenteric venule permeability differently in males and females. Further studies are needed to differentiate the permeability responses with respect to age and gender.

Intravascular metastatic cancer cell homotypic aggregation at the sites of primary attachment to the endothelium.

The two major theories of cancer metastasis, the seed and soil hypothesis and the mechanical trapping theory, view tumor cell adhesion to blood vessel endothelia and cancer cell aggregation as corresponding key components of the metastatic process. Here, we demonstrate in vitro, ex vivo, and in vivo that metastatic breast and prostate carcinoma cells form multicellular homotypic aggregates at the sites of their primary attachment to the endothelium. Our results suggest that metastatic cell heterotypic adhesion to the microvascular endothelium and homotypic aggregation represent two coordinated subsequent steps of the metastatic cascade mediated largely by similar molecular mechanisms, specifically by interactions of tumor-associated Thomsen-Friedenreich glycoantigen with the beta-galactoside-binding protein, galectin-3. In addition to inhibiting neoplastic cell adhesion to the endothelium and homotypic aggregation, disrupting this line of intercellular communication using synthetic Thomsen-Friedenreich antigen masking and Thomsen-Friedenreich antigen mimicking compounds greatly affects cancer cell clonogenic survival and growth as well. Thus, beta-galactoside-mediated intravascular heterotypic and homotypic tumor cell adhesive interactions at the sites of a primary attachment to the microvascular endothelium could play an important role during early stages of hematogenous cancer metastasis.