Oscillatory shear alters endothelial hydraulic conductivity and nitric oxide levels

This study addresses the role of nitric oxide (NO) and downstream signaling pathways in mediating the influences of oscillatory shear stress on the hydraulic conductivity (L(p)) of bovine aortic endothelial cell (BAEC) monolayers. Exposure of BAEC monolayers to 20 dyne/cm2 steady shear stress for 3 h induced a 3.3-fold increase in L(p). When an oscillatory shear amplitude of 10 dyne/cm2 was superimposed on a steady shear of 10 dyne/cm2 to produce a non-reversing oscillatory shear pattern (10+/-10 dyne/cm2), L(p) increased by 3.0-fold within 90 min. When the amplitude was increased to 15 dyne/cm2, resulting in a reversing oscillatory shear pattern (10+/-15 dyne/cm2), the increase in L(p) over 3 h was completely suppressed. Twenty and 10+/-10 dyne/cm2 induced 2.9- and 2.6-fold increases in NO production above non-sheared controls, respectively, whereas 10+/-15 dyne/cm2 stimulated a 14-fold increase in NO production. The inhibition of L(p) with reversing oscillatory shear may be associated with alterations in cyclic guanosine monophosphate (cGMP) production downstream of NO which is up-regulated by reversing oscillatory shear, but is unaffected by steady shear.     

Evidence for a role of hyaluronan in the spacing of fibrils within collagen bundles in rabbit synovium

Synovial hydraulic resistance is vital for the retention of intra-articular fluid, and originates within the matrix of biopolymers in the intercellular gaps. Specific digestion of hyaluronan resulted in a increase in synovial hydraulic permeability from 0.478+/-0.24 microl min(-1) cm H(2)O(-1) in control tissue to 4.561+/-0.40 microl min(-1) cm H(2)O(-1) (mean+/-S.D., n=6 rabbits, P<0.001 t test). To investigate whether hyaluronidase also altered the interstitial ultrastructure, morphometry of hyaluronidase treated synovium was carried out. The most striking novel finding was that hyaluronidase treatment reduced extrafibrillar volume fraction within the synovial collagen bundles from 50.5+/-11.1% to 36.8+/-15.5% (mean+/-S.D., n=6 rabbits, P<0.001, two-way anova). This was accompanied by a reduction in interfibrillar centre to centre spacing from 101+/-11 (control) to 84+/-6 nm (mean+/-S.D.; n=6 rabbits, P<0.001) in enzyme-treated bundles. Individual fibrils showed a small but highly significant reduction in cross-sectional diameter from 76.9+/-6.3 to 72.5+/-6.3 nm (mean+/-S.E.; P<0.001) after hyaluronidase treatment. The findings indicate that hyaluronan chains have a major organisational role within the collagen bundle itself. The trans-synovial pathway comprises bundles and substantial areas of intervening, bundle-free matrix, and it is possible that bundle collapse contributes to a rise in overall permeability by increasing the inter-bundle space.     

Hyaluronan digestion and synthesis in an experimental model of metastatic tumour

To approach the question of hyaluronan catabolism in tumours, we have selected the cancer cell line H460M, a highly metastatic cell line in the nude mouse. H460M cells release hyaluronidase in culture media at a high rate of 57 pU/cell/h, without producing hyaluronan. Hyaluronidase was measured in the H460M cell culture medium at the optimum pH 3.8, and was not found above pH 4.5, with the enzyme-linked sorbent assay technique and zymography. Tritiated hyaluronan was digested at pH 3.8 by cells or cell membranes as shown by gel permeation chromatography, but no activity was recorded at pH 7 with this technique. Hyaluronan was digested in culture medium by tumour slices, prepared from tumours developed in nude mice grafted with H460M cells, showing that hyaluronan could be digested in complex tissue at physiological pH. Culture of tumour slices with tritiated acetate resulted in the accumulation within 2 days of radioactive macromolecules in the culture medium. The radioactive macromolecular material was mostly digested by Streptomyces hyaluronidase, showing that hyaluronan was its main component and that hyaluronan synthesis occurred together with its digestion. These results demonstrate that the membrane-associated hyaluronidase of H460M cells can act in vivo, and that hyaluronan, which is synthesised by the tumour stroma, can be made soluble and reduced to a smaller size by tumour cells before being internalised and further digested.     

The influence of ovarian steroids on ovine endometrial glycosaminoglycans

The ovine endometrium is subjected to cyclic oscillations of estrogen and progesterone in preparation for implantation. One response to fluctuating hormonal levels is the degree of hydration of the tissue, suggesting cyclical alterations in glycosaminoglycan/proteoglycan content. The aim of the present study was to quantitate and characterize glycosaminoglycans in the ovine endometrium during estrogen and progesterone dominant stages. Endogenous endometrial glycosaminoglycan content was determined by chemical analysis and characterized by enzyme specific or chemical degradation. [(35)S]-sulphate and [(3)H]-glucosamine labeled proteoglycans/glycosaminoglycans were extracted by cell lysis or with 4M guanidine-HCl. Extracts were purified by anion exchange and gel chromatography and characterized as above. Estrogen and progesterone dominant endometrium contained 3.2 +/- 0.1 and 2.1 +/- 0.1 mg endogenous glycosaminoglycan/g dehydrated tissue, respectively. Characterization of endogenous glycosaminoglycan showed chondroitin sulphate and hyaluronan contributing over 80%. The major difference between hormonal dominant tissue was a higher estrogenic hyaluronan percentage and a higher progestational keratan sulphate percentage (p < 0.001). Estrogen dominant tissue incorporated 1.6-1.9 fold more radiolabeled proteoglycans/glycosaminoglycans (p < 0.001). Analysis of newly synthesized proteoglycans/glycosaminoglycans revealed a heparan/chondroitin sulphate ratio of 1:2.2-2.5. Keratan sulphate was not detected. Estrogenic hyaluronan was 1.6 fold greater in [(3)H]-labeled tissue. Analysis of labeled proteoglycans/glycosaminoglycans revealed two size classes with apparent molecular weights >2.0 x 10(6) and 0.8-1.1 x 10(5) and a charge class eluting between 0.1-0.5 M NaCl. The greater glycosaminoglycan content (particularly hyaluronan) and synthesis in estrogen dominant tissue supports a role for steroid hormones in endometrial glycosaminoglycan/proteoglycan regulation and consequent tissue hydration. It also suggests a role for these macromolecules in endometrial function and possibly the implantation process.     

A novel method for measuring membrane conductance changes by a voltage-sensitive optical probe.

This study presents a method whose principles enable using a voltage-sensitive optical probe, to quantitatively measure conductivity changes elicited in membrane vesicles and cells. The procedure is based on the fact that the amplitude of the transmembrane potential difference, established across a membrane by an external electric field, is decreased when membrane conductivity is increased upon incorporation of ionophores into the membrane. The method was applied to osmotically swollen thylakoid membranes whose membrane conductivity was changed by the addition of gramicidin or ionomycin. The electric field induced stimulated luminescence from photosystem I (electrophotoluminescence-EPL) was used as a voltage-sensitive optical probe. We calculated the induced conductance changes by using a calibrated EPL vs external electric field response curve and measuring the ionophore-mediated attenuation of the EPL signal. The calculated ionophore-unmodified conductance of the thylakoid membrane yields a value of 171 +/- 56 nS/cm. The value of the membrane conductance, modified by 10 nM gramicidin was found to be 190 +/- 56 nS/cm. The modified membrane conductance and the membrane conductance changes induced by 1 microM ionomycin in the presence of CaCl2 were found to be 186 +/- 3 nS/cm and 15 +/- 3 nS/cm, respectively.     

Mitral inertance in humans: critical factor in Doppler estimation of transvalvular pressure gradients

The pressure-velocity relationship across the normal mitral valve is approximated by the Bernoulli equation DeltaP = 1/2 rhoDeltav(2) + M. dv/dt, where DeltaP is the atrioventricular pressure difference, rho is blood density, v is transmitral flow velocity, and M is mitral inertance. Although M is indispensable in assessing transvalvular pressure differences from transmitral flow, this term is poorly understood. We measured intraoperative high-fidelity left atrial and ventricular pressures and simultaneous transmitral flow velocities by using transesophageal echocardiography in 100 beats (8 patients). We computed mean mitral inertance (M) by M = integral((DeltaP)-(1/2 x rho v(2))dt/integral(dv/dt)dt and we assessed the effect of the inertial term on the transmitral pressure-flow relation. ranged from 1.03 to 5.96 g/cm(2) (mean = 3.82 +/- 1.22 g/cm(2)). DeltaP calculated from the simplified Bernoulli equation (DeltaP = 1/2. rhov(2)) lagged behind (44 +/- 11 ms) and underestimated the actual peak pressures (2.3 +/- 1.1 mmHg). correlated with left ventricular systolic pressure (r = -0.68, P < 0.0001) and transmitral pressure gradients (r = 0.65, P < 0.0001). Because mitral inertance causes the velocity to lag significantly behind the actual pressure gradient, it needs to be considered when assessing diastolic filling and the pressure difference across normal mitral valves.     

Red cell extensional recovery and the determination of membrane viscosity.

A theory of membrane viscoelasticity developed by Evans and Hochmuth in 1976 is used to analyze the time-dependent recovery of an elongated cell. Before release, the elongated cell is the static equilibrium where external forces are balanced by membrane elastic force resultants. Upon release, the cell recovers its initial shape with a time-dependent exponential behavior characteristic of the viscoelastic solid model. It is shown that the model describes the time-dependent recovery process very well for a time constant in the range of 0.1-0.13 s. The time constant is the ratio membrane surface viscosity eta:membrane surface elasticity mu. Measurements for the shear modulus mu of 0.006 dyne/cm give a value for the surface viscosity of red cell membrane as a viscoelastic solid material of eta = mu tc = (6-8) X 10(-4) poise . cm.     

Cover Image,Volume 116, Number 5, May 2019

Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) present an attractive alternative to primary EC sources for vascular grafting. However, there is a need to mature them towards either an arterial or venous subtype. A vital environmental factor involved in the arteriovenous specification of ECs during early embryonic development is fluid shear stress; therefore, there have been attempts to employ adult arterial shear stress conditions to mature hPSC-ECs. However, hPSC-ECs are naïve to fluid shear stress, and their shear responses are still not well understood. Here, we used a multiplex microfluidic platform to systematically investigate the dose-time shear responses on hPSC-EC morphology and arterial-venous phenotypes over a range of magnitudes coincidental with physiological levels of embryonic and adult vasculatures. The device comprised of six parallel cell culture chambers that were individually linked to flow-setting resistance channels, allowing us to simultaneously apply shear stress ranging from 0.4 to 15 dyne/cm ². We found that hPSC-ECs required up to 40 hr of shear exposure to elicit a stable phenotypic change. Cell alignment was visible at shear stress <1 dyne/cm ², which was independent of shear stress magnitude and duration of exposure. We discovered that the arterial markers NOTCH1 and EphrinB2 exhibited a dose-dependent increase in a similar manner beyond a threshold level of 3.8 dyne/cm ², whereas the venous markers COUP-TFII and EphB4 expression remained relatively constant across different magnitudes. These findings indicated that hPSC-ECs were sensitive to relatively low magnitudes of shear stress, and a critical level of ~4 dyne/cm ² was sufficient to preferentially enhance their maturation into an arterial phenotype for future vascular tissue engineering applications.     

Effects of electric charge on hydraulic conductivity of pulmonary interstitium

The hydraulic conductivity of pulmonary interstitium was measured in a short isolated segment of interstitium surrounding a large pulmonary artery (1-3 mm diam) of the rabbit. The flow rate of the following solutions was measured sequentially: normal saline, polycation protamine sulfate (0.08 mg/ml), cationic dextran (0.1 or 1.5%) or anionic dextran (0.1 or 1.5%), and hyaluronidase (testes, 0.02%) solution. The pH of all solutions was adjusted to 7.35-7.40. The ratios of the flow of protamine sulfate and cationic dextran to that of saline averaged 2.3 +/- 0.92 (SD, n = 7) and 3.0 +/- 1.2 (n = 6), respectively. The anionic dextran-to-saline flow ratio averaged 0.72 +/- 0.28 (n = 13). Flow increased in the presence of positively charged molecules and decreased in the presence of negatively charged molecules. At a lower pH of 5.0-6.0, only 0.1% cationic dextran had an effect on interstitial conductivity. Thus pulmonary interstitium at physiological pH has the properties of a negatively charged membrane. The increased interstitial conductivity caused by the positively charged molecules was not observed after treatment with hyaluronidase. These effects of electric charge on interstitial conductivity were partly attributed to the presence in the interstitium of negatively charged hyaluronan.     

Regulation of vocal fold transepithelial water fluxes.

Vocal fold hydration is critical to phonation. We hypothesized that the vocal fold generates bidirectional water fluxes, which are regulated by activity of the Na(+)-K(+)- ATPase. Western blots and immunohistochemistry demonstrated the presence of the alpha-subunit Na(+)-K(+)-ATPase in the canine vocal fold (n = 11). Luminal cells, basal and adjacent one to two layers of suprabasal cells within stratified squamous epithelium, were immunopositive, as well as basolateral membranes of submucosal seromucous glands underlying transitional epithelia. Canine (n = 6) and ovine (n = 14) vocal fold mucosae exhibited transepithelial potential differences of 8.1 +/- 2.8 and 9.3 +/- 1.3 mV (lumen negative), respectively. The potential difference and short-circuit current (ovine = 31 +/- 4 microA/cm(2); canine = 41 +/- 10 microA/cm(2)) were substantially reduced by luminal administration of 75 microM acetylstrophanthidin (P < 0.05). Ovine (n = 7) transepithelial water fluxes decreased from 5.1 +/- 0.3 to 4.3 +/- 0.3 microl x min(-1) x cm(-2) from the basal to luminal chamber and from 5.2 +/- 0.2 to 3.9 +/- 0.3 microl x min(-1) x cm(-2) from the luminal to basal chamber by luminal acetylstrophanthidin (P < 0.05). The presence of the Na(+)-K(+)-ATPase in the vocal fold epithelium and the electrolyte transport derived from its activity provide the intrinsic mechanisms to regulate cell volume as well as vocal fold hydration.     

Density and hydration of fresh and fixed human skeletal muscle

The maximum tetanic tension of skeletal muscle (P(0)) is often estimated based on calculation of physiological cross-sectional area (PCSA). PCSA depends on muscle volume, pennation angle, and fiber length. Studies documenting PCSA in fixed human muscles usually compute muscle volume by dividing muscle mass by density. These studies use a density value of 1.0597 g/cm(3), which was originally based on unfixed rabbit and canine muscle tissue. Due to the dehydration effects of different fixation methods, the variable hydration that occurs when fixed tissue is stored in buffered saline, and the potential for species-specific muscle density, this value may be incorrect and an accurate value for fixed human muscle density is needed. To obtain an accurate density and water content values, 4% formaldehyde-fixed (n=54) and 37% formaldehyde-fixed (n=54) cadaveric human muscle samples were divided into 6 groups (0, 6, 12, 18, 24, or 30 h) for hydration in phosphate buffered saline (PBS). Measurements of volume, water content, and mass were made enabling calculation of muscle density. Additionally, water content was measured in living muscle (n=4) to determine the appropriate hydration time in PBS. Comparisons among groups demonstrated a significant increase in muscle water content and muscle volume over time, reaching living tissue levels after 24h, but, interestingly, the hydration process did not affect muscle density. These data yield a density value (mean+/-SE) of 1.112+/-0.006 g/cm(3) in 4% formaldehyde-fixed muscle and 1.055+/-0.006 g/cm(3) in 37% formaldehyde-fixed muscle. These results indicate that the use of inappropriate hydration times or density values can produce PCSA errors of 5-10%.     

Work of adhesion of respiratory tract mucus.

A method was devised to measure the work of adhesion (WA) to a substrate of mucus, a viscoelastic gel, from the measured contact angle of glycerol on a mucus substrate and the known physical properties of a Teflon surface. Fifteen sputum samples from cystic fibrosis (CF) patients were compared with 25 mucus samples from canine tracheal pouches (CP), studied in the hydrated and partially dehydrated states. Apparent viscosity (eta A) and recoverable shear strain (SR) were measured by fluxgate magnetometry, and water content was inferred from vapor pressure osmometry. Na+, K+, and Ca2+ concentrations were measured with specific ion electrodes and Cl- with a chloridimeter. The Cl- concentration of the CP mucus was inversely proportional to its osmolality, and the Cl- concentration of the CP mucus was 102.5 +/- 1.6 meq/l compared with 55.6 +/- 2.5 meq/l for CF sputum. When CP mucus osmolality was increased from 316.0 +/- 5.5 to 430.0 +/- 7.5 mosmol/kg, WA increased from 25.1 +/- 1.8 to 31.1 +/- 1.2 ergs/cm2 and eta A increased from 391 +/- 55 to 622 +/- 121 P, respectively. CF sputum WA was 30.2 +/- 0.6 ergs/cm2, eta A was 1,110 +/- 316 P, and osmolality was 466.0 +/- 14.0 mosmol/kg. The increased WA and eta A of mucus in CF patients may thus be dependent on the hydration of mucus, which is related to the documented Cl- transport defect.     

Osmotic water permeability of human red cells

The osmotic water permeability of human red cells has been reexamined with a stopped-flow device and a new perturbation technique. Small osmotic gradients are used to minimize the systematic error caused by nonlinearities in the relationship between cell volume and light scattering. Corrections are then made for residual systematic error. Our results show that the hydraulic conductivity, Lp, is essentially independent of the direction of water flow and of osmolality in the range 184-365 mosM. the mean value of Lp obtained obtained was 1.8 +/- 0.1 (SEM) X 10-11 cm3 dyne -1 s-1.     

Effects of fluid shear stress on mRNA expression of carbonic anhydrase II in polarized rat osteoclasts

The present study was designed to determine the effects of fluid shear stress on the mRNA expression of carbonic anhydrase II (CAII) in polarized rat osteoclasts. Cellular morphology of the polarized osteoclasts generated by a mechanical anatomical technique was examined by tartrate-resistant acid phosphatase (TRAP) staining and the osteoclastic resorption of dentine slices. The polarized osteoclasts were then stress-loaded by using a flow shear stress device newly developed by the osteoclast research group (patent number 200420034438; China), at 9 dyne/cm(2) for various time periods [0 (control group), 15, 30, 60, and 120 min], or at various stress levels [0 (control), 0.9, 2.9, 8.7, and 26.3 dyne/cm(2)] for 30 min. The mRNA expression of CAII was quantified using real-time fluorescent quantitative PCR (RT-PCR) and the data were analyzed with SPSS 12.0 software. The polarized osteoclasts were larger than regular monocytes (about 30 microm diameter) with irregular configuration, and the majority of polarized osteoclasts appeared to be spherical and had approximately 2-20 nuclei. The TRAP positive polarized osteoclasts showed asymmetrical red staining in the cytoplasm, and had many filaments and vacuoles. These cells formed resorptive pits in dentine slices. The levels of CAII mRNA expression were shown to be time-dependent, with the E+5 copy numbers being 7.88+/-0.09, 11.14+/-0.12, 15.83+/-0.18, 1.94+/-0.02, and 1.37+/-0.01 in cells treated at 9 dyne/cm(2) for 0, 15, 30, 60 and 120 min, respectively (P < 0.05). The levels of CAII mRNA expression (E+5 copy numbers) in cells treated with the stress levels of 0, 0.9, 2.9, 8.7 and 26.3 dyne/cm(2) were 7.97+/-0.201, 11.26+/-0.688, 15.94+/-0.201, 31.88+/-1.496, and 45.08+/-2.639, respectively (P < 0.05). These results indicate that there is a relationship between the fluid shear stress and the mRNA expression of CAII in polarized rat osteoclasts.     

Electrical properties of rabbit corneal endothelium as determined from impedance measurements.

Alternating- and direct-current electrical characteristics of rabbit corneal endothelium were studied under varying experimental conditions. The measurements were performed by sending a 10-microA current (AC or DC) across the tissue layer. Maximal values of transendothelial potential difference and resistance were 1.3 +/- 0.1 mV and 73 +/- 6 omega . cm2, respectively. The short-circuit current was estimated from the potential and resistance values. Impedance loci were obtained for the frequency range 0.5-100 kHz. A capacitive reactance (C = 0.63 +/- 0.02 microF/cm2) was observed in the 100 Hz-100 kHz range. To relate the impedance data to the electrical parameters of the cell membranes, the voltage-divider ratio was determined by sending square pulse across the tissue and measuring voltage responses across the apical and basal membranes with an intracellular microelectrode. The intracellular potential difference was on the average -61 +/- 1 mV, and the voltage-divider ratio was found to be between 0.33 and 4. Impedance data were fit by a computer to an equivalent circuit representing a "lumped" model, and the agreement between the model and the data was satisfactory. The results are discussed in terms of both the morphological characteristics and properties of the fluid transport mechanism across the preparation.     

Local heating of human skin by millimeter waves: effect of blood flow

We investigated the influence of blood perfusion on local heating of the forearm and middle finger skin following 42.25 GHz exposure with an open ended waveguide (WG) and with a YAV mm wave therapeutic device. Both sources had bell-shaped distributions of the incident power density (IPD) with peak intensities of 208 and 55 mW/cm(2), respectively. Blood perfusion was changed in two ways: by blood flow occlusion and by externally applied vasodilator (nonivamide/nicoboxil) cream to the skin. For thermal modeling, we used the bioheat transfer equation (BHTE) and the hybrid bioheat equation (HBHE) which combines the BHTE and the scalar effective thermal conductivity equation (ETCE). Under normal conditions with the 208 mW/cm(2) exposure, the cutaneous temperature elevation (DeltaT) in the finger (2.5 +/- 0.3 degrees C) having higher blood flow was notably smaller than the cutaneous DeltaT in the forearm (4.7 +/- 0.4 degrees C). However, heating of the forearm and finger skin with blood flow occluded was the same, indicating that the thermal conductivity of tissue in the absence of blood flow at both locations was also the same. The BHTE accurately predicted local hyperthermia in the forearm only at low blood flow. The HBHE made accurate predictions at both low and high perfusion rates. The relationship between blood flow and the effective thermal conductivity (k(eff)) was found to be linear. The heat dissipating effect of higher perfusion was mostly due to an apparent increase in k(eff). It was shown that mm wave exposure could result in steady state heating of tissue layers located much deeper than the penetration depth (0.56 mm). The surface DeltaT and heat penetration into tissue increased with enlarging the irradiating beam area and with increasing exposure duration. Thus, mm waves at sufficient intensities could thermally affect thermo-sensitive structures located in the skin and underlying tissue.     

A microfabricated array bioreactor for perfused 3D liver culture

We describe the design, fabrication, and performance of a bioreactor that enables both morphogenesis of 3D tissue structures under continuous perfusion and repeated in situ observation by light microscopy. Three-dimensional scaffolds were created by deep reactive ion etching of silicon wafers to create an array of channels (through-holes) with cell-adhesive walls. Scaffolds were combined with a cell-retaining filter and support in a reactor housing designed to deliver a continuous perfusate across the top of the array and through the 3D tissue mass in each channel. Reactor dimensions were constructed so that perfusate flow rates meet estimated values of cellular oxygen demands while providing fluid shear stress at or below a physiological range (<2 dyne cm(2)), as determined by comparison of numerical models of reactor fluid flow patterns to literature values of physiological shear stresses. We studied the behavior of primary rat hepatocytes seeded into the reactors and cultured for up to 2 weeks, and found that cells seeded into the channels rearranged extensively to form tissue like structures and remained viable throughout the culture period. We further observed that preaggregation of the cells into spheroidal structures prior to seeding improved the morphogenesis of tissue structure and maintenance of viability. We also demonstrate repeated in situ imaging of tissue structure and function using two-photon microscopy.     

Trophoblast migration under flow is regulated by endothelial cells

During pregnancy, trophoblasts enter the uterine vasculature and are found in spiral arteries far upstream of uterine capillaries. It is unknown whether trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by intravasation directly into spiral arteries after interstitial migration. We have developed an in vitro system consisting of early gestation macaque monkey trophoblasts cocultured with uterine endothelial cells and have exposed the cells in a parallel plate flow chamber to physiological levels of shear stress. Videomicroscopy followed by quantitative image analysis revealed that the migratory activity (expressed as average displacement and average migration velocity) of trophoblasts cultured on top of endothelial cells remained unchanged between shear stresses of 1-30 dyne/cm(2) whereas activity of trophoblasts alone increased with increasing shear stress. When the direction of migration was assessed at 1 and 7.5 dyne/cm(2), the extent of migration against and with flow was roughly equal for both trophoblasts alone and cocultured trophoblasts. At shear stress levels of 15 and 30 dyne/cm(2), trophoblasts incubated alone showed a significant decrease in migration against flow and corresponding increased migration in the direction of flow. In contrast, trophoblasts cocultured with uterine endothelial cells maintained the same extent of migration against flow at all shear stress levels. Migration against flow was also maintained when trophoblasts were cultured with endothelial cell-conditioned medium or fixed endothelial cells. The results indicate that factors expressed on the surface of uterine endothelial cells and factors released by endothelial regulate trophoblast migration under flow.     

Evaluation of radius of gyration and intrinsic viscosity molar mass dependence and stiffness of hyaluronan

Nine hyaluronan (HA) samples were fractionated by size-exclusion chromatography, and molar mass (M), radius of gyration (Rg), and intrinsic viscosity ([eta]) were measured in 0.15 M NaCl at 37 degrees C by on-line multiangle light scattering and viscometer detectors. Using such method, we investigated the Rg and [eta] molar mass dependence for HA over a very wide range of molar masses: M ranging from 4 x 10(4) to 5.5 x 10(6) g/mol. The Rg and the [eta] molar mass dependence found for HA showed a meaningful difference. The Rg = f(M) power law was substantially linear in the whole range of molar masses explored with a constant slope of 0.6. In contrast, the [eta] = f(M) power law (Mark-Houwink-Sakurada plot) showed a marked curve shape, and a linear regression over the whole range of molar masses does not make sense. Also the persistence length (stiffness) for HA was estimated. The persistence length derived by using both the Odijk's model (7.5 nm from Rg vs M data) and the Bohdanecky's plot (6.8 nm from [eta] vs M data) were quite similar. These persistence length values are congruent with a semistiff conformation of HA macromolecules.     

Effects of albumin, dextran, and hyaluronidase on pulmonary interstitial conductivity

The fluid conductivity of albumin solutions of various concentrations relative to that of saline was measured in the interstitium surrounding a short segment of a large (1.5- to 3-mm-diam) blood vessel of an isolated rabbit lung of which air spaces and vasculature were filled with silicon rubber. At a constant driving pressure, the flow of the following solutions was measured sequentially: normal saline and albumin solution (3, 5.5, 8, or 15 g/100 ml saline), hyaluronidase solution (0.02 g/100 ml), and albumin solution (same concentration used before hyaluronidase solution). The albumin-to-saline flow ratios averaged 1.00 +/- 0.23 (SD), 1.01 +/- 0.21, 1.32 +/- 0.63, and 1.54 +/- 0.36 for albumin concentrations of 3, 5.5, 8, and 15 g/100 ml, respectively. These ratios were higher than the corresponding values of 0.88, 0.78, 0.72, and 0.5 expected if the flow of albumin solution were to depend only on fluid viscosity. The flow of dextran and hyaluronan solutions was more viscosity dependent than the flow of albumin solutions. The increased flow of albumin solution could be the result of a reduced excluded volume of albumin caused by collagen and glycosaminoglycans with an increased albumin concentration. The flow of hyaluronidase solution was 24 +/- 22 (SD)-fold (n = 36) larger than the flow of albumin solution. Thus hyaluronan was responsible for most of the hydraulic resistance of the interstitium to bulk flow. After its degradation, the flow of albumin solution became more viscosity dependent. The interaction between plasma proteins and glycosaminoglycans in the pulmonary interstitium could serve to enhance clearance of microvascular filtrate, particularly under conditions of large protein leaks.