Virginia H. Huxley

Fluorescent Dyes Modify Properties of Proteins Used in Microvascular Research

Objective: Fluorescent dyes, used frequently to label proteins for microvascular experiments, are assumed to not alter the proteins physicochemical characteristics. We tested the validity of that assumption for two probes, bovine serum albumin (BSA) and α‐lactalbumin.

Evidence of porcine and human endothelium activation by cancer-associated carbohydrates expressed on glycoproteins and tumour cells

It is well established that after metastatic cancer cells escape the primary tumour and enter the circulation, their interactions with microvascular endothelium of a target organ constitute an essential rate‐limiting step in haematogenous cancer metastasis. However, the physiological and biochemical processes supporting neoplastic cell arrest and retention in the microcirculation are still poorly understood. In this study, we present experimental evidence that microvascular endothelium of metastasis‐prone tissues undergoes activation in response to desialylated cancer‐associated carbohydrate structures such as Thomsen–Friedenreich (TF) antigen (Galβ1–3GalNAc) expressed on circulating glycoproteins and neoplastic cells. The metastasis‐associated endothelium activation, manifested by marked increase in endothelial cell surface galectin‐3 expression, causes gradual decrease in cancer cell velocities (from 72 × 102± 33 × 102μm s−1 to 7.6 × 102± 1.9 × 102μm s−1, mean ±s.d.) accompanied by a corresponding increase in the percentage of rolling cells (from 3.3%± 1.2% to 24.3%± 3.6%, mean ±s.d.), and results in human breast and prostate carcinoma cell arrest and retention in the microvasculature. This process, which could be of high importance in haematogenous cancer metastasis, was inhibited efficiently by an anti‐TF antigen function‐blocking antibody. Carbohydrate‐mediated endothelial activation could be a process of physiological significance as it probably occurs in the interactions between a variety of circulating constituents and the vessel wall.

Leakage responses to L-NAME differ with the fluorescent dye used to label albumin

Nitric oxide synthase (NOS) inhibitors have been reported to increase as well as to decrease microvascular transport of macromolecules in a variety of models. This study was performed to determine whether the influence of NOS inhibition on albumin leakage was dependent on the fluorescent dyes used to label albumin. Albumin leakage was assessed in rat mesenteric venules during control conditions and after exposure to the NOS inhibitor N G-nitro-l-arginine methyl ester (l-NAME). Albumin was labeled with any one of four dyes: FITC, sulforhodamine 101 [Texas Red (TR)], dichlorotriazinyl aminofluorescein (DTAF), or Oregon Green 514 (OG). Superfusion withl-NAME (10-4 M) was accompanied by an increase in leakage of FITC-labeled albumin ( n = 12) but not of albumin labeled with DTAF ( n = 10), TR ( n = 10), or OG ( n = 4). In vessels perfused with both FITC- and TR-labeled albumin ( n = 12), superfusion with l-NAME increased leakage of FITC- but not TR-labeled albumin. In conclusion, albumin leakage responses tol-NAME differ among various fluorescent dyes. Therefore, caution is advised in comparison of albumin leakage results that utilize different fluorescent dyes.

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 (PRSAs) 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 PRSAs would not differ significantly. Supporting our hypothesis, the median collecting lymphatic PRSAs (3.5 ± 1.0 × 10−7 cm s−1, N= 22) did not differ significantly from the median venular PRSAs (4.0 ± 1.0 × 10−7 cm s−1, N= 8, P= 0.61). For collecting lymphatics the diffusive permeability (Pd= 2.5 × 10−7 cm s−1) was obtained from the relationship of apparent PRSAs and pressure. While the measured PRSAs, Pd 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.

Continuous real time ex vivo epifluorescent video microscopy for the study of metastatic cancer cell interactions with microvascular endothelium

Recent studies suggest that only endothelium-attached malignant cells are capable of giving rise to hematogenous cancer metastases. Moreover, tumor cell adhesion to microvascular endothelium could be crucial in metastasis predilection to specific organs or tissues. However, the existing in vitro and in vivo techniques do not provide for sufficient delineation of distinct stages of a dynamic multi-step intravascular adhesion process. Here we report the development of an experimental system allowing for prolonged continuous ex vivo real-time observation of malignant cell adhesive interactions with perfused microvessels of a target organ in the context of its original tissue. Specifically, the vasculature of excised dura mater perfused with prostate cancer cells is described. An advantage of this technique is that selected fluorescently labeled tumor cells can be followed along identified vascular trees across the entire tissue specimen. The techniques provide for superior microvessel visualization and allow for uninterrupted monitoring and video recording of subsequent adhesion events such as rolling, docking (initial reversible adhesion), locking (irreversible adhesion), and flattening of metastatic cancer cells within perfused microvasculature on a single cell level. The results of our experiments demonstrate that intravascular adhesion of cancer cells differs dramatically from such of the leukocytes. Within dura microvessels perfused at physiological rate, non-interacting, floating, tumor cells move at velocities averaging 7.2×103 μm/s. Some tumor cells, similarly to leukocytes, exhibit rolling-like motion patterns prior to engaging into more stable adhesive interactions. In contrast, other neoplastic cells became stably adhered without rolling showing a rapid reduction in velocity from 2×103 to 0 μm/s within fractions of a second. The experimental system described herein, while developed originally for studying prostate cancer cell interactions with porcine dura mater microvasculature, offers great flexibility in adhesion experiments design and is easily adapted for use with a variety of other tissues including human.

Lymphatic fluid: exchange mechanisms and regulation

Abstract  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.

Differential Coronary Microvascular Exchange Responses to Adenosine: Roles of Receptor and Microvessel Subtypes

Objective: To assess the role of adenosine receptors in the regulation of coronary microvascular permeability to porcine serum albumin (PsPSA).

Gas Diffusion through the Fractal Landscape of the Lung: How Deep Does Oxygen Enter the Alveolar System?

We investigate oxygen transport to and across alveolar membranes in the human lung, the last step in the chain of events that takes oxygen through the bronchial airways to the peripheral, acinar airways. This step occurs by diffusion. We carry out analytic and numerical computations of the oxygen current for fractal, space-filling models of the acinus, based on morphological data of the acinus and appropriate values for the transport constants, without adjustable parameters. The computations address the question whether incoming oxygen reaches the entire available membrane surface (reaction-limited, unscreened oxygen current), a large part of the surface (mixed reaction/diffusion-limited, partly screened current), or only the surface near the entrance of the acinus (diffusion-limited, completely screened current). The analytic treatment identifies the three cases as sharply delineated screening regimes and finds that the lung operates in the partial-screening regime, close to the transition to no screening, for respiration at rest; and in the no-screening regime for respiration at exercise. The resulting currents agree well with experimental values. We test the analytic treatment by comparing it with numerical results for two-dimensional acinus models and find very good agreement. The results provide quantitative support for the conclusion, obtained in other work, that the space-filling fractal architecture of the lung is optimal with respect to active membrane surface area and minimum power dissipation. At the level of the bronchial tree, we show that the space-filling architecture provides optimal slowing down of the airflow from convection in the bronchial airways to diffusion in the acinar airways.

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 hormones released into the bloodstream when heart muscle is stretched (e.g. zero‐gravity, hypertension, congestive heart failure) and serve to reduce the blood volume. •  One way that these peptides relieve blood volume is to increase the permeability of the smallest blood vessels, facilitating fluid and protein distribution into the tissue spaces. •  Whether these peptides target lymphatic vessel function to participate in fluid distribution is currently unknown. •  ANP and BNP (100 nm) both elicited significant increases in lymphatic vessel permeability, but altered contractile function differentially in vivo. •  A likely consequence is that more fluid leaks from the lymphatics into the tissues, which represents a novel compensation for volume overload. This work demonstrates for the first time that lymphatic vessel permeability can be regulated in vivo.