Human and porcine aortic valve endothelial and interstitial cell isolation and characterization.

Background: Calcific aortic valve stenosis (AVS) is defined by pathological changes in the aortic valve (AV) and their predominant cell types: valvular interstitial (VICs) and endothelial cells (VECs). Understanding the cellular and molecular mechanisms of this disease is a prerequisite to identify potential pharmacological treatment strategies. In this study, we present a unique aortic valve cell isolation technique to acquire specific human and porcine cell populations and compared VICs and VECs of these species with each other for the first time. Methods: AV cells were isolated from tissue obtained from human patients undergoing surgical aortic valve replacement (SAVR) or from porcine hearts. Functional analysis and in vitro experiments revealed that endothelial-to-mesenchymal transition (EndMT) can be induced in hVECs, leading to a significant increase in mesenchymal markers. In vitro calcification experiments of VICs demonstrated pronounced expression of calcification markers and visible calcific deposits in Alizarin Red staining in both species after incubation with pro-calcific media. Results: Cells isolated from patient-derived AVs showed mesenchymal and endothelial-specific gene signatures (VIC and VEC, respectively). For instance, von Willebrand factor (vWF) and platelet endothelial adhesion molecule-1 (PECAM1) were upregulated in VECs, while the myofibroblastic markers alpha-smooth muscle actin (α-SMA) and vimentin (VIM) were downregulated in VECs compared to VICs. Analysis of cell function by migration revealed that VECs are more migratory than VICs. Induction of EndMT in vitro in VECs displayed increased expression of EndMT markers and decreased expression of endothelial markers, confirming their mesenchymal transdifferentiation ability. In vitro calcification of VICs revealed upregulation of alkaline phosphatase (ALPL), a hallmark of calcification. In addition, other calcification-related genes such as osteocalcin (BGLAP) and runt-related factor 2 (RUNX2) were upregulated. Alizarin red staining of calcified cells provided a further layer of confirmation that the isolated cells were VICs with osteoblastic differentiation capacity. Conclusion: This study aims to take a first step towards standardizing a reproducible isolation technique for specific human and porcine VEC and VIC populations. A comparison of human and porcine aortic valve cells demonstrated that porcine cells may serve as an alternative cellular model system in settings where human tissue is difficult to obtain.

Expression of the familial cardiac valvular dystrophy gene, filamin-A, during heart morphogenesis.

Myxoid degeneration of the cardiac valves is a common feature in a heterogeneous group of disorders that includes Marfan syndrome and isolated valvular diseases. Mitral valve prolapse is the most common outcome of these and remains one of the most common indications for valvular surgery. While the etiology of the disease is unknown, recent genetic studies have demonstrated that an X-linked form of familial cardiac valvular dystrophy can be attributed to mutations in the Filamin-A gene. Since these inheritable mutations are present from conception, we hypothesize that filamin-A mutations present at the time of valve morphogenesis lead to dysfunction that progresses postnatally to clinically relevant disease. Therefore, by carefully evaluating genetic factors (such as filamin-A) that play a substantial role in MVP, we can elucidate relevant developmental pathways that contribute to its pathogenesis. In order to understand how developmental expression of a mutant protein can lead to valve disease, the spatio-temporal distribution of filamin-A during cardiac morphogenesis must first be characterized. Although previously thought of as a ubiquitously expressed gene, we demonstrate that filamin-A is robustly expressed in non-myocyte cells throughout cardiac morphogenesis including epicardial and endocardial cells, and mesenchymal cells derived by EMT from these two epithelia, as well as mesenchyme of neural crest origin. In postnatal hearts, expression of filamin-A is significantly decreased in the atrioventricular and outflow tract valve leaflets and their suspensory apparatus. Characterization of the temporal and spatial expression pattern of filamin-A during cardiac morphogenesis is a crucial first step in our understanding of how mutations in filamin-A result in clinically relevant valve disease.

Characterization of rat and human steroid sulfatases.

Rat and human steroid sulfatases were purified from liver and placenta, respectively, by the same procedure. The rat and human enzymes were solubilized with Triton X-100, and purified by immunoaffinity chromatography with a monoclonal antibody showing high binding activities to both the enzymes. They were further purified by high-pressure anion-exchange chromatography to compare their structural and catalytic properties. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that both enzymes had a molecular weight of 62,000. The enzymes had similar amino acid compositions and amino-terminal amino acid sequences. Significant differences of the optimum pH, Michaelis constant and maximum velocity were observed between these enzymes. The optimum pH of each enzyme varied from 6.0 to 8.0, depending on substrates and with or without Triton X-100. In detergent-free media, steroid sulfates competitively inhibited the ability of these enzymes to hydrolyze 4-nitrophenyl sulfate. In media containing Triton X-100, on the other hand, the inhibition types of the steroid sulfates on the hydrolyzing activities of the rat and human enzymes were noncompetitive- and mixed-types, respectively.

Ultrastructural localization of arylsulfatase C activity in rat kidney.

Metal precipitation techniques for ultrastructural demonstration of arylsulfatase C activity were studied in rat kidney. Possible substrates for the techniques were biochemically tested with regard to their velocity of enzymatic hydrolysis and their specificity for arylsulfatase C. Effects of buffers and capturing metals were also examined. The results of these biochemical studies were then verified histochemically. Incubation in a medium containing 1 mM 4-methylumbelliferyl sulfate, 1% barium chloride, 0.1 M imidazole-HCl buffer (pH 7.5), and 5% sucrose achieved identifiable results in adequately fixed kidney. Precipitation of barium sulfate was localized mainly in the endoplasmic reticulum and perinuclear cisterns of the epithelial cells in the descending portions of proximal tubules.

A monoclonal antibody to rat liver arylsulfatase C and its application in immunohistochemistry.

We purified arylsulfatase C from rat liver microsomes and prepared a monoclonal antibody (P42C2) to the purified enzyme. By SDS-PAGE and immunoblotting analysis using P42C2, the molecular weight of the purified enzyme and of the enzyme in liver and kidney microsomes were estimated at 62,000 daltons. P42C2 caused little inhibition of arylsulfatase C activity, and was bound only slightly to liver microsomes. Localization of arylsulfatase C was studied at the light and electron microscopic level by the indirect immunoperoxidase method using P42C2. In rat liver, arylsulfatase C was detected mainly in the hepatocytes, and less frequently in endothelial cells, Kupffer's cells, and Ito's cells. In rat kidney, strong staining was observed in the straight portions of the proximal tubules. The podocytes, interstitial cells, endothelial cells, and epithelial cells of Henle's thin limbs were stained faintly. By electron microscopy, arylsulfatase C was found localized on the membranes of the endoplasmic reticulum and nuclear envelopes in these cells. These immunohistochemical findings agree with the localization demonstrated by an enzyme-histochemical method which we had previously developed.

Somatic mosaicism for a DMD gene deletion.

Mosaicism is a mixed state, with two cell populations of different genetic origins caused by a cell mutation occurring after fertilization. In the present case, DNA analysis of lymphocytes led to a DMD diagnosis before death. Postmortem immunocytochemical and DNA analysis showed somatic mosaicism. At age 18 years, blood lymphocyte DNA analysis showed a DMD gene deletion, upstream from exon 7 to the 5' end containing both muscle and brain promoters. As the patient's mother and elder sister had no deletions, he was considered to have a new mutation. Immunocytochemical studies of postmortem tissues showed that dystrophin was absent from the tongue, deltoid, intercostal, psoas and rectus femoris muscles, but there was a mix of dystrophin-positive and negative fibers in the rectus abdominis, cardiac, temporalis and sternocleidomastoid muscles. All diaphragm cells were dystrophin positive. Polymerase chain reaction (PCR) amplification from all tissues except the temporalis and sternocleidomastoid muscles, diaphragm and kidney, in which no deletion was found, showed the deletion from at least exon 6 to the 5' end containing both muscle and brain promoters. In this case, a genomic deletion of the DMD gene contributed to the formation of tissues derived from both ectoderm and endoderm, and cells of mesodermal origin showed genotypic and phenotypic heterogeneity. Our results indicate a mutation of the present case may have occurred just before the period of germ layer formation.

Characterization of a monoclonal antibody to hog thyroid peroxidase and its use for immunohistochemical localization of the peroxidase in the thyroid gland.

A monoclonal antibody (30.1.2) to hog thyroid peroxidase was produced, purified, and characterized. The IgG of 30.1.2 formed an immune complex with the peroxidase in a 1:2 or 1:1 molar ratio depending on the IgG to antigen ratio in the incubation mixture. Immune complex formation did not inhibit the peroxidase activity, which was actually activated 2-fold in the 1:1 complex. Studies of the binding of the conjugate of the IgG or its Fab' with horseradish peroxidase to untreated and acetone-treated thyroid microsomes showed that the IgG conjugate could bind to only a very small portion of the total binding sites (thyroid peroxidase) present in untreated microsomes even after prolonged incubation. The binding of the Fab' conjugate to untreated microsomes, on the other hand, increased as the incubation time was increased, reaching 40% of the total sites after 20 h of incubation. These findings indicated that thyroid peroxidase is localized on the inner surface of the microsomal membranes and that the Fab' conjugate, but not the IgG conjugate, can slowly penetrate through the membrane barrier to reach the peroxidase. Immunohistochemical experiments using the Fab' conjugate as a probe revealed that most thyroid peroxidase in the thyroid gland is located in the endoplasmic reticulum and perinuclear cisternae of the follicular cell, although a small amount could occasionally be detected in the apical membrane including microvilli. In contrast to previous reports, no thyroid peroxidase could be found in other cellular structures such as Golgi apparatus and apical vesicles by the immunohistochemical technique employed.

A simple method for cryopreservation of purified adult pig pancreatic cells.

Cryopreservation of islet cells may benefit many aspects of islet transplantation including storage, transport of islets, immunomodulation, reduction of the exocrine contaminants. Isolation and purification of islets from large mammalian pancreases have been encountered by many problems. We reported an autodigestion method for preparation of adult pig pancreatic cells. In this paper, we describe a newly invented simple method for cryopreservation of purified adult pig pancreatic cells. The viability and function of islet cells before (precryo.) and after (postcryo.) the cryopreservation procedure were compared. Islet cell harvested by the autodigestion method were suspended in cryoprotectant "Cell banker." A biofreezing vessel "Bicell" containing a cryogenic vial with cell suspension was frozen at -80 degrees C. After 4 wk, the cryogenic vial was rapidly thawed at 37 degrees C water, the islet cells were washed and cultured for overnight. Number of dithizone-stained precryo. and postcryo. cells were 1.71 +/- 0.5 x 10(5) and 1.75 +/- 0.98 x 10(5) cells/pancreas respectively; therefore, the viability was 99%. Insulin release following high and low concentrations of glucose in precryo. (12.02 +/- 3.63 ng/ml: low-glucose, 9.81 +/- 3.59 ng/ml: high glucose) and precryo. (5.57 +/- 4.03 ng/ml: low-glucose, 10.24 +/- 8.88 ng/ml: high glucose) cells showed a marked improvement in response in postcryo. cells. The insulin content of precryo. and postcryo. cells were 914.4 +/- 277.4 and 93.6 +/- 15.4 ng/ml, respectively. This simple and efficient cryopreservation protocol may be applied for a large-scale storage of adult pig islet cells to establish an islet bank for transplantation.

Regional distribution of arylsulfatase C and estrone-sulfate sulfatase activities in rat brain and hypophysis.

Regional distributions of arylsulfatase C and estrone-sulfate sulfatase activities were studied in rat brain and hypophysis by both histochemical and biochemical methods. Both methods showed that high activities of both enzymes were localized in pineal gland, choroid plexus, and adenohypophysis. Ultracytochemical techniques visualized the arylsulfatase C activity in the endoplasmic reticulum and the nuclear envelope of pineal cells, ependymal cells, and some types of cells of the adenohypophysis.

A simple method for purification of adult pig pancreatic endocrine cells.

Adult pig pancreatic endocrine cells were harvested by autodigestion without added enzymes. The isolated, crude cells were purified by mono-poly resolving medium (MPRM). The purity of the harvested cells was determined by dithizone staining and the number of pancreatic endocrine cells was counted. A large number of the cells were stained red with dithizone and showed a high viability and a good insulin secretory response to glucose stimulation. The average number of cells purified by MPRM was 3.40 +/- 1.32 x 10(5) cells/g pancreas and the number of dithizone-stained cells was 2.81 +/- 1.09 x 10(5) cells/g pancreas. The insulin secretion from the pancreatic endocrine cells was maintained throughout a 40-day observation period and high glucose stimulation induced an increase in insulin secretion from the cultured cells. In the cells purified by MPRM, light and electron microscopic studies showed the cells to be typical pancreatic endocrine cells. The present purification method using MPRM allowed us quickly to obtain a large amount of adult pig pancreatic endocrine cells from the unpurified preparations. It is thought to be useful for transplantation and biochemical or biological studies of adult pig pancreatic endocrine cells.

Fluctuation of fluorescent material in the rat's and human leucocytes under various situation.

When modified FALCK-HILLARP technic was applied to blood smears of rat and human specimens, the fluorescence was detected not only in platelets, but in leucocytes. In rat leucocytes and platelets, the bimodal daily rhythm of fluorescent material is observed under physiological condition. The rhythm is modified considerably if animals are exposed to the continuous lighting. The intensity and number of the fluorescent cells in the human smears increased markedly in patients suffering from schizophrenia compared with controls. The content of fluorescent material in leucocytes assumes to be closely related with an activity of the living states.

[Immunohistochemical analysis of the normal and cystic kidney using antibody against polycystin 2].

A second gene responsible for polycystic kidney disease(PKD) has been identified recently, and an antisera(YCC2) against this gene's product, polycystin 2, has been generated. In the present study, we investigated the normal distribution of polycystin 2 in human adult kidneys and analyzed the expression of polycystin 2 in the cystic tubules of kidneys from patients with PKD and acquired cystic disease of the kidney(ACDK). The expression of polycystin 2 in normal regions of resected human kidneys, 4 cases of autosomal dominant polycystic kidney disease(ADPKD) and 4 cases of ACDK was examined by immunohistochemically staining the specimens with a polyclonal antibody specific to the C-terminal region of polycystin 2. This region is specific to polycystin 2 and does not crossreact with polycystin 1. In normal kidneys, prominent expression of polycystin 2 was observed in the distal tubules. A faint level of expression was detected in the proximal tubules, and the glomerulus and vessels were almost negative for expression. In the cystic kidneys of ADPKD patients, 68.7% of the cystic tubules stained positively for YCC2, although partial staining was seen in 41.2% of the positive cystic tubules. Although the genetic background of the samples is unknown, the co-existence of positive and negative cysts suggest that a "two-hit" hypothesis is feasible and that the mutations are likely to be missense or in frame changes. In ACDK cysts, YCC2-positive staining was prominent in small cysts (less than 0.5 mm in diameter), which were also positive for DBA, a marker for distal tubules. In contrast, larger cysts of more than 0.5 mm in diameter which stained positive for a proximal tubule marker, Lotus T, tended to be less positively stained for YCC2. Overall, 94.5% of the cysts stained positive for YCC2, which is a much higher rate than that of PKD cysts. These results suggest that ACDK cysts may be generated by a different mechanism from that of PKD cysts.

Mechanism of elevated local oxidant stress in early anti-glomerular basement membrane nephritis: an evaluation of oxidant production and superoxide dismutase expression.

The present study was designed to identify the mechanism of increased oxidant stress in the rat model of anti-glomerular basement membrane nephritis. Sixty-three Sprague-Dawley rats were injected with nephrotoxic serum and evaluated 1 to 24 hours later. In these rats, CeCl3 deposition, an index of hydrogen peroxide production, was observed on the surfaces of glomerular endothelial cells and polymorphonuclear leukocytes, whereas no such depositions were observed in controls. Renal cortical level of lipid peroxidation products (phosphatidylcholine hydroperoxide) was significantly (p < 0.05) elevated at one hour after the injection and remained elevated at least for 24 hours. Protein levels of glomerular Mn-superoxide dismutase (SOD) decreased from 1.55 +/- 0.38 microgram/mg protein to 0.67 +/- 0.18 microgram/mg protein at one hour and normalized by 12 hours after the injection. The activity of the enzyme showed a similar trend. In contrast, Mn-SOD mRNA increased 3.4-fold at 3 hours after the injection. In situ hybridization showed increased Mn-SOD mRNA expression in glomeruli. Cu/Zn-SOD mRNA expression was transiently suppressed. These results indicated that both increase in local production of reactive oxygen species (ROS) and reduction in antioxidant enzyme activities are responsible for the enhanced oxidant stress in the heterologous phase of anti-glomerular basement membrane nephritis. The paradoxical increase in Mn-SOD mRNA expression indicates that the posttranscriptional down regulation of Mn-SOD (i.e., reduction in protein and activity) and the increased ROS may activate transcription of the gene.

Formation of coronary arteries sprouting from the primitive aortic sinus wall of the chick embryo.

The formation of coronary arteries in chick embryos was observed by scanning electron microscopy on injected casts as well as by transmission electron microscopy. Usually, 2-4 primitive coronary arteries appear from the right aortic sinus below the level of the cusp margin, and 1-3 from the left one. As development proceeds, the arteries are generally reduced in number to form a single definitive coronary artery on each side. Canalization of the arteries seems to take place by partially degenerative changes of the primordia.

Changes of profiles of neutrophil granulocytes induced with the administration of L-DOPA and prednisolone.

Morphological changes of granulocytes following combined treatment with L-DOPA and prednisolone were elucidated at the electron microscopic level. Neutrophil granulocytes exhibited marked changes in their surface as evidenced by scanning electron microscope. It is suggested that neutrophil granulocytes are sensitive to some biogenic amines.

Formation of the myelinated nerve fiber layer in the chicken retina.

Oligodendrocytes in the ganglion cell layer, the myelinating cells in the chicken retina, were investigated morphologically and quantitatively. Oligodendroblasts divided in the inner retinal layer around the 14th day of incubation and differentiated into oligodendrocytes. The oligodendrocytes started sheathing an axon in the nerve fiber layer at the 14th day of incubation. The number of myelin lamellae increased rapidly during the first week after chicks had hatched. An immunological reaction of anti-myelin basic protein was observed on the myelin sheaths in the nerve fiber layer and on the oligodendrocytes in the ganglion cell layer. These results suggest that the oligodendrocytes form the myelinated nerve fiber layer of the chicken and that they act independently of the Müller cells during myelination.

Tridimensional observation of fluorescent granular perithelial (FGP) cells in rat cerebral blood vessels.

The tridimensional appearance and distribution of FGP (fluorescent granular perithelial) cells was studied by means of light and scanning electron microscopy. In young rats they first appeared as hexagonal cells in that were closely associated; later they transformed into slender forms and were loosely arranged. Scanning electron microscope observation gave a general view of FGP cells, their globular vacuolated inclusions, and their hypertrophied protrusion into the luminal surface of blood vessels. The nodular protrusions may be related to the limitation of blood flow in small cerebral blood vessels.

Anomalous looping, atrioventricular cushion dysplasia, and unilateral ventricular hypoplasia in the mouse embryos with right isomerism induced by retinoic acid.

BACKGROUND: Visceroatrial heterotaxy syndrome is characterized by abnormality of visceral laterality and complex cardiovascular anomalies usually involving both the outflow and inflow tract. Morishima et al. (1995) showed that mouse embryos treated with all-trans retinoic acid at embryonic day 6.5 (primitive streak stage) induces this syndrome. METHODS: To investigate the morphogenetic process of visceroatrial heterotaxy syndrome, we examined retinoic acid-treated mouse embryos at embryonic days 9-15 using scanning electron microscopy. RESULTS: The sinoatrial connection was first distinguished for the determination of situs as early as at embryonic day 10.5. Normal visceroatrial situs was found in 57% of all treated embryos, and the rest had abnormal situs, in which right isomerism was found in 81%. In the right-isomeric mouse, the cardiac morphology was characterized by abnormal looping together with dysplasia of the inflow and outflow tract cushion; that is, the primitive right ventricle was usually deviated cranially to various degrees, the atrioventricular cushion appeared trilobed in a half of them, and unilateral ventricular hypoplasia was noted in about one-third of them after embryonic day 14.5. CONCLUSIONS: An anomalous relation between the atrioventricular cushions and the interventricular septum appeared to have caused a restrictive inflow to the unilateral ventricle, leading to ventricular chamber hypoplasia on the ipsilateral side. Thus, we clarified that retinoic-acid treatment at the primitive streak stage disturbed cardiac looping and formation of atrioventricular cushion development, which secondarily influenced ventricular chamber development.

Ultrastructural changes in rat palatal epithelium after beta-aminopropionitrile.

Beta-Aminopropionitrile (BAPN) retarded or suppressed epithelial changes in the medial edge of the palatal process in later stages of gestation in rats. Programmed cell death did not follow the usual pattern, and only a few lysosomes were observed on day 18 of gestation. The sensitivity of the medial epithelium to BAPN appeared to be different in various areas of the palatal epithelium; the epithelium on the anterior region of the palatal process was hypertrophied and keratinized, while posteriorly the medial or neighboring epithelium was very thin and, in neonatal rats, the covering was absent. A basal lamina was distinct in the anterior region and indistinct or fragmented posteriorly. Collagen fibers did not develop adjacent to the basal lamina, and an amorphous material was scattered throughout the mesenchymal tissue. These findings suggest that BAPN decreases the "connecting capacity" between mesenchyme and epithelium, and results in a modification of epithelial changes.

Evidence for the possible function of the fluorescent granular perithelial cells in brain as scavengers of high-molecular-weight waste products.

The fluorescent granular perithelium (FGP) of rats and humans under experimental and pathological conditions was examined with the electron microscope. The FGP incorporated high molecular-weight protein (ferritin) and carbon particles administered intraventricularly. In a case of spontaneous cerebral hemorrhage, the FGP was found to contain lipoidal products and minute fragmented cell debris. The FGP in a patient with lipidosis contained pale inclusion bodies. In aged individuals, the inclusion bodies formed irregular larger aggregates.