Generation of Femtosecond Laser-Cut Decellularized Corneal Lenticule Using Hypotonic Trypsin-EDTA Solution for Corneal Tissue Engineering

Purpose. To establish an optimized and standardized protocol for the development of optimal scaffold for bioengineering corneal substitutes, we used femtosecond laser to process human corneal tissue into stromal lenticules and studied to find the most efficient decellularization method among various reagents with different tonicities. Methods. The decellularization efficacy of several agents (0.1%, 0.25%, and 0.5% of Triton X-100, SDS, and trypsin-EDTA (TE), resp.) with different tonicities was evaluated. Of all protocols, the decellularization methods, which efficiently removed nuclear materials examined as detected by immunofluorescent staining, were quantitatively tested for sample DNA and glycosaminoglycan (GAG) contents, recellularization efficacy, and biocompatibilities. Results. 0.5% SDS in hypertonic and isotonic buffer, 0.25% TE in hypotonic buffer, and 0.5% TE in all tonicities completely decellularized the corneal lenticules. Of the protocols, decellularization with hypotonic 0.25 and 0.5% TE showed the lowest DNA contents, while the GAG content was the highest. Furthermore, the recellularization efficacy of the hypotonic TE method was better than that of the SDS-based method. Hypotonic TE-treated decellularized corneal lenticules (DCLs) were sufficiently transparent and biocompatible. Conclusion. We generated an ideal protocol for DCLs using a novel method. Furthermore, it is possible to create a scaffold using a bioengineered corneal substitute.

Abstract 343: Endothelial Regulator of G-Protein Signaling-5 Maintains Endothelial Permeability and Controls Vascular Smooth Muscle Cell Migration in vitro

GAP-inhibitor, RGS5 has been described as a VSMC marker and is associated with plaque stability. Its role in endothelial cells (EC) is unclear. We examined RGS5 expression patterns in human vasculature and hypothesized that loss of RGS5 alters EC biology and crosstalk with VSMC such, that it could promote atherogenesis. Methods: Human internal mammary artery (IMA), saphenous vein (SV) and failing myocardium (MYO) samples were stained for RGS5, αSMA and vWF. RGS5 or scrambled siRNA knocked down (RGS5-KD) HUVECs were used. oxLDL or TNFα induced THP1-adhesion to HUVECs was assessed. The 2h diffusion rate of Evans-blue bound to 0.1%-BSA/DPBS through a HUVEC monolayer was spectrophotometrically measured in the presence of PTX. HUVECs were also subjected to spheroid sprouting in collagen/methylcellulose. qRT-PCR using the Δ(ΔCT) method with HPRT as house-keeper was used to assess MMP-9, VEGF-A and caveolin-1 expression in transfected cells. To test RGS5-dependent paracrine effects of EC-secreted factors on VSMC, isotonic buffer was conditioned for 2h by transfected HUVECs (CB). CB was added to VSMC during a scratch assay. Results: RGS5 signal was enhanced in the endothelium of SV compared to IMA (63±9 vs 22±4 %vessel perimeter, p<0.05, n=4). Similar patterns were observed in myocardium. In IMA plaques RGS5 was prominently reduced in medial VSMC underlying and in the endothelium covering the plaque compared to non-diseased vessels. No effect on TNFα- or oxLDL-induced adhesion was observed in RGS5-KD HUVECs. HUVEC permeability increased in RGS5-KD cells (1 vs 2.5± 0.2 normalized OD, n=4, p<0.05), which was reduced by PTX (1.4±0.25). MMP-9 (1.0±0.03 vs 2.5±0.54, p<0.05, n=5), VEGF-A (1.0±0.02 vs 2.8±0.76, p<0.05, n=5) and Caveolin-1 (1.0±0.01 vs 1.6±0.17, n=3) expression (2-Δ (ΔCT)) were increased in RGS5-KD HUVECs. RGS5-KD increased the number of spheroid sprouts (2.1± 0.5 vs 3.5± 0.3,n=4, p<0.05). CB of RGS5-KD HUVECs increased VSMC migration (49±2.2 vs 67±2.6 %coverage, p<0.05, n=7). Conclusion: In conclusion, RGS5 levels could be involved in vascular differentiation. Its effects on permeability and VSMC migration point towards a prominent role in angiogenesis and atherogenesis. Atherosclerosis studies in RGS5-deficient animals are warranted.

Sperm quality assessments for endangered razorback suckers Xyrauchen texanus

Flow cytometry (FCM) and computer-assisted sperm motion analysis (CASA) methods were developed and validated for use with endangered razorback suckersXyrauchen texanuscollected (n=64) during the 2006 spawning season. Sperm motility could be activated within osmolality ranges noted during milt collections (here 167–343 mOsm/kg). We hypothesized that sperm quality of milt collected into isoosmotic (302 mOsm/kg) or hyperosmotic (500 mOsm/kg) Hanks' balanced salt solution would not differ. Pre-freeze viabilities were similar between osmolalities (79%±6 (s.e.m.) and 76%±7); however, post-thaw values were greater in hyperosmotic buffer (27%±3 and 12%±2;P=0.0065), as was mitochondrial membrane potential (33%±4 and 13%±2;P=0.0048). Visual estimates of pre-freeze motility correlated with total (r=0.7589; range 23–82%) and progressive motility (r=0.7449) by CASA and were associated with greater viability (r=0.5985;P<0.0001). Count (FCM) was negatively correlated with post-thaw viability (r=−0.83;P=0.0116) and mitochondrial function (r=−0.91;P=0.0016). By FCM-based assessments of DNA integrity, whereby increased fluorochrome binding indicated more fragmentation, higher levels were negatively correlated with count (r=−0.77;P<0.0001) and pre-freeze viabilities (r=−0.66;P=0.0004). Fragmentation was higher in isotonic buffer (P=0.0234). To increase reproductive capacity of natural populations, the strategy and protocols developed can serve as a template for use with other imperiled fish species, biomonitoring, and genome banking.

Zinc inhibits cAMP-stimulated Cl secretion via basolateral K-channel blockade in rat ileum

Zn, an essential micronutrient and second most abundant trace element in cell and tissues, reduces stool output when administered to children with acute diarrhea. The mechanism by which Zn improves diarrhea is not known but could result from stimulating Na absorption and/or inhibiting anion secretion. The aim of this study was to investigate the direct effect of Zn on intestinal epithelial ion absorption and secretion. Rat ileum was partially stripped of serosal and muscle layers, and the mucosa was mounted in lucite chambers. Potential difference and short-circuit current were measured by conventional current-voltage clamp method.86Rb efflux and uptake were assessed for serosal K channel and Na-K-2Cl cotransport activity, respectively. Efflux experiments were performed in isolated cells preloaded with86Rb in the presence of ouabain and bumetanide, whereas uptake experiments were performed in low-Cl isotonic buffer containing Ba and ouabain. Neither mucosal nor serosal Zn affected glucose-stimulated Na absorption. In contrast, forskolin-induced Cl secretion was markedly reduced by serosal but not mucosal addition of Zn. Zn also substantially reversed the increase in Cl secretion induced by 8-bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP) with half-maximal inhibitory concentration of 0.43 mM. In contrast, serosal Zn did not alter Cl secretion stimulated by carbachol, a Ca-dependent agonist. Zn inhibited 8-BrcAMP-stimulated86Rb efflux but not carbachol-stimulated86Rb efflux. Zn had no effect on bumetanide-sensitive86Rb uptake, Na-K-ATPase, or CFTR. We conclude from these studies that Zn inhibits cAMP-induced Cl secretion by blocking basolateral membrane K channels.

The plasma-membrane Ca2+-ATPase of Leishmania donovani is an extrusion pump for Ca2+

Controlled exposure of Leishmania donovanipromastigotes to hypotonic shock results in the formation of deflagellated unsealed ghosts of original polarity that largely retain the pellicular microtubular structure associated with plasma membrane of the parasite. Gentle shearing followed by suspension of the purified membrane in appropriate isotonic buffer containing Mg2+ (4 mM) results in the formation of sealed everted vesicles. The presence of Mg2+ (4 mM) appears to be essential for efficient sealing and also to prevent leakiness. ATP-dependent Ca2+ accumulation can be demonstrated in these vesicles. Km values for Ca2+ and ATP were 125 nM and 0.8 mM respectively. The accumulated Ca2+ reaches a concentration of 1.1 mM. Ca2+ uptake is completely inhibited by vanadate (40 ƁM) and several thiol-modifying agents. Using 5,5ƀ-dithiobis-(2-nitrobenzoic acid) as the modifying agent, an excellent correlation between loss of enzyme activity and transport capability and their parallel regeneration in the presence of 2 mM dithiothreitol was demonstrated. Using 2ƀ,7-bis(carboxyethyl)-5(6)-carboxyfluorescein as the fluorescent pH probe, it was observed that Ca2+ entry into the vesicles is accompanied by an outward movement of H+ from the vesicles. Taken together, this paper establishes that the high-affinity transmembrane Ca2+-ATPase [Ghosh, Ray, Sarkar and Bhaduri (1990) J. Biol. Chem. 265, 11345Ő11351; Majumdar, Mukherjee, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440Ő18446] is an extrusion pump for Ca2+ in this human pathogen.

Kinetics and osmoregulation of Na(+)-and Cl(-)-dependent betaine transporter in rat renal medulla

Betaine is one of the major organic osmolytes that accumulate in the renal medulla in response to high extracellular tonicity. Recent studies in MDCK cells have shown that betaine is taken up by an Na(+)- and Cl(-)-dependent transporter located on the basolateral membrane. We demonstrate here the presence of Na(+)-Cl(-)-dependent betaine transporter(s) in tubule suspensions prepared from the rat outer and inner medulla. The betaine transport activity was two to three times higher in the inner medulla compared with the outer medulla. The removal of Na+ and Cl- reduced betaine uptake in the outer medullary tubules by 86% and 82%, respectively. The betaine uptake was decreased by 39% in hypotonic buffer (189 mosmol/ kgH2O) and increased by 82% in hypertonic buffer (545 mosmol/kgH2O), compared with isotonic buffer (308 mosmol/ kgH2O). Kinetic studies of Na(+)-dependent betaine uptake in the outer medullary tubules revealed both a low- and a high-affinity component as follows: low-affinity and high volume component with Michaelis constant (K(m)1) of 8.6 mM and maximal uptake rate (Vmax1) of 112 pmol.microgram protein-1.h-1; and a low-volume and high-affinity component with K(m)2 of 0.141 mM and Vmax2 of 10 pmol. microgram protein-1.h-1. To investigate whether the Na(+)-Cl(-)-dependent betaine transporter is regulated by tonicity in vivo, we quantitated its mRNA in rat renal cortex and outer and inner medulla using both canine and rat Na(+)-Cl(-)-dependent betaine transporter cDNA probes. A single band of 3.0 kb was seen in the Northern blots prepared from both outer and inner medulla, but not in the cortex. Water deprivation for 3 days increased the abundance of this mRNA in the outer and inner medulla by 140% and 170%, respectively, but did not affect its expression in the cortex. In conclusion, Na(+)-Cl(-)-dependent betaine transporter(s) is present in rat outer and inner medullary tubules, and betaine transporter mRNA abundance is regulated by the hydration state in vivo.

Regulation of cell volume and [Ca2+]i in attached human fibroblasts responding to anisosmotic buffers

Alteration of the cell volume of attached fibroblasts with anisosmotic buffers was used to examine the relationship between cell membrane perturbation and intracellular Ca2+ concentration ([Ca2+]i) and to study pathways that may be involved in transducing this response. Human periodontal ligament gingival fibroblasts grown on cover slips were loaded with fura 2-acetoxymethyl ester. The relative cell volume change of single fibroblasts was estimated by measurement of fluorescence intensity at the isosbestic wavelength (356 nm), and [Ca2+]i was calculated from ratiometric fura 2 emission with excitation at 345 and 380 nm. Isotonic buffer (300 mosmol/kgH2O) was substituted with either hypertonic (600 mosmol/kgH2O) or hypotonic (150 mosmol/kgH2O) buffer after baseline recordings. Attached cells exhibited a rapid decrease in cell volume and [Ca2+]i after hypertonic buffer treatment, which was associated with an increase in filamentous actin staining. In contrast, cells treated with hypotonic buffers demonstrated an increase in cell volume (mean approximately 10%), a significant decrease in filamentous actin staining, and a rapid transient elevation in [Ca2+]i (mean approximately 280 nM). This [Ca2+]i rise was significantly inhibited by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, gadolinium ions (P < 0.05), and inhibitors of actin assembly. These results indicate that [Ca2+]i fluxes in response to hypotonic cell swelling in attached fibroblasts are mediated by stretch-activated ion channels and are dependent on actin filaments.

Investigation of the subcellular location of the tetrapyrrole-biosynthesis enzyme coproporphyrinogen oxidase in higher plants

The subcellular location of two enzymes in the biosynthetic pathway for protoporphyrin IX, coproporphyrinogen (coprogen) oxidase (EC 1.3.3.3) and protoporphyrinogen (protogen) oxidase (EC 1.3.3.4) has been investigated in etiolated pea (Pisum sativum) leaves and spadices of cuckoo-pint (Arum maculatum). Plant tissue homogenized in isotonic buffer was subjected to subcellular fractionation to prepare mitochondria and plastids essentially free of contamination by other cellular organelles, as determined by marker enzymes. Protogen oxidase activity measured fluorimetrically was reproducibly found in both mitochondria and etioplasts. In contrast, coprogen oxidase could be detected only in etioplasts, using either a coupled fluorimetric assay or a sensitive radiochemical method. The implications of these results for the synthesis of mitochondrial haem in plants is discussed.

Transport of sodium chloride and water in rat ascending vasa recta

Experiments were undertaken to test the hypothesis that transcapillary small solute (NaCl and urea) gradients drive water across ascending vasa recta (AVR). Axial gradients of NaCl and urea were eliminated with furosemide. AVR were perfused with buffer containing fluorescein isothiocyanate-labeled dextran and 22Na. Perfusion of AVR with isotonic buffer at 10 and 20 nl/min yielded collectate-to-perfusate 22Na ratios of 0.17 +/- 0.05 and 0.34 +/- 0.03, respectively, in AVR of 601 +/- 56 and 583 +/- 46 microns mean length, respectively. A 22Na permeability of 113.2 +/- 12.8 x 10(-5) cm/s was determined. AVR were perfused at 20 nl/min with buffer NaCl of 0 (hypotonic to papilla), 161 (isotonic), or 500 mM (hypertonic). Transcapillary volume flux was not significantly different in these groups (3.8 +/- 1.5, 4.6 +/- 1.5, and 2.1 +/- 1.4 nl.min-1.mm-1, respectively). AVR were perfused in the hydropenic kidney at 5 nl/min antegrade from tip to base and retrograde from base to tip, which was a maneuver designed to impose physiological transcapillary NaCl and urea gradients of opposite direction. Volume fluxes were -1.4 +/- 0.05 and -1.3 +/- 0.04 nl.min-1.mm-1 in these groups, respectively. These data demonstrate that the AVR are highly permeable to NaCl and that physiological small solute gradients do not influence water movement across the AVR wall.