Interstitial Cell Population Research Articles

Comparison of analytical and inverse finite element approaches to estimate cell viscoelastic properties by micropipette aspiration

The viscoelastic properties of cells are important in predicting cell deformation under mechanical loading and may reflect cell phenotype or pathological transition. Previous studies have demonstrated that viscoelastic parameters estimated by finite element (FE) analyses of micropipette aspiration (MA) data differ from those estimated by the analytical half-space model. However, it is unclear whether these differences are statistically significant, as previous studies have been based on average cell properties or parametric analyses that do not reflect the inherent experimental and biological variability of real experimental data. To determine whether cell material parameters estimated by the half-space model are significantly different from those predicted by the FE method, we implemented an inverse FE method to estimate the viscoelastic parameters of a population of primary porcine aortic valve interstitial cells tested by MA. We found that inherent differences between the analytical and inverse FE estimation methods resulted in statistically significant differences in individual cell properties. However, in cases with small pipette to cell radius ratios and short loading periods, model-dependent differences were masked by experimental and cell-to-cell variability. Analytical models that account for finite cell-size and loading rate further relaxed the experimental conditions for which accurate cell material parameter estimates could be obtained. These data provide practical guidelines for analysis of MA data that account for the wide range of conditions encountered in typical experiments.

Ano1 is a selective marker of interstitial cells of Cajal in the human and mouse gastrointestinal tract

Populations of interstitial cells of Cajal (ICC) are altered in several gastrointestinal neuromuscular disorders. ICC are identified typically by ultrastructure and expression of Kit (CD117), a protein that is also expressed on mast cells. No other molecular marker currently exists to independently identify ICC. The expression of ANO1 (DOG1, TMEM16A), a Ca(2+)-activated Cl(-) channel, in gastrointestinal stromal tumors suggests it may be useful as an ICC marker. The aims of this study were therefore to determine the distribution of Ano1 immunoreactivity compared with Kit and to establish whether Ano1 is a reliable marker for human and mouse ICC. Expression of Ano1 in human and mouse stomach, small intestine, and colon was investigated by immunofluorescence labeling using antibodies to Ano1 alone and in combination with antibodies to Kit. Colocalization of immunoreactivity was demonstrated by epifluorescence and confocal microscopy. In the muscularis propria, Ano1 immunoreactivity was restricted to cells with the morphology and distribution of ICC. All Ano1-positive cells in the muscularis propria were also Kit positive. Kit-expressing mast cells were not Ano1 positive. Some non-ICC in the mucosa and submucosa of human tissues were Ano1 positive but Kit negative. A few (3.2%) Ano1-positive cells in the human gastric muscularis propria were labeled weakly for Kit. Ano1 labels all classes of ICC and represents a highly specific marker for studying the distribution of ICC in mouse and human tissues with an advantage over Kit since it does not label mast cells.

Dose‐dependent effects on cell proliferation, seminiferous tubules, and male germ cells in the fetal rat testis following exposure to di(n‐butyl) phthalate

Adult male rats gestationally exposed to di(n-butyl)phthalate (DBP) have dysgenetic testes characterized by seminiferous epithelial degeneration, clustering of Leydig cells, and decreased spermatogenesis. Cell proliferation and apoptosis are key processes regulating development of the testis, and alterations in these processes may underlie testicular dysgenesis. To determine whether gestational exposure to DBP affects cell proliferation and apoptosis in the developing rat testis. Pregnant dams were exposed to different dose levels of DBP in mid-gestation and cellular outcomes in fetal and early postnatal testes were assayed by histological and morphometric approaches. Gestational exposure to high dose DBP inhibited proliferation of fetal testicular somatic cells but did not affect apoptosis. Exposed fetal testes had a smaller volume and decreased cell numbers, with decreases in both the tubular and interstitial cell populations. A reduction was observed in the testis volume and altered seminiferous tubule morphometry at > or =50 mg/kg/d, and a decreased testicular cell number at > or =30 mg/kg/d DBP. The number of multinucleated gonocytes in DBP-exposed fetal testes increased after exposure to > or =100 mg/kg/d. The number of proliferating cells in the DBP-exposed testis rapidly rose after birth (when exposure stopped), and the testis volume and the total cell number was comparable to control by postnatal day 2. DBP reversibly inhibits proliferation of somatic cells in the fetal rat testis. Decreased proliferation, rather than increased apoptosis, is the underlying mechanism of altered fetal development of DBP-exposed seminiferous tubules contributing to testicular dysgenesis.

Phenotypic alteration of interstitial cells of Cajal in idiopathic sigmoid megacolon

Interstitial cells of Cajal (ICCs) are detected as a pacemaker of gastrointestinal movement and express c-kit and CD34. Recently, ICCs have implicated pathogenesis in several human diseases presenting gastrointestinal motor dysfunction. This study was performed to clarify the role of ICCs in idiopathic sigmoid megacolon using histological and immunohistochemical examinations. Four adult patients with idiopathic sigmoid megacolon and 11 controls were studied. Histology and immunocytochemistry using NSE, S100, c-kit, and CD34 were performed in conjunction with quantitative analysis using the public domain NIH image program. Little histological change in neuromuscular structures in megacolon was observed. Immunohistochemistry demonstrated remarkable decrease of c-kit expressing ICCs without reduction of CD34 expression in the similar interstitial cell population. This observation was further supported by quantitative assessment using public domain NIH image program. A specific downregulation of c-kit in ICCs may be a cause of idiopathic sigmoid megacolon in adults.

Cofilin is a marker of myofibroblast differentiation in cells from porcine aortic cardiac valves

The formation of myofibroblasts in valve interstitial cell (VIC) populations contributes to fibrotic valvular disease. We examined myofibroblast differentiation in VICs from porcine aortic valves. In normal valves, cells immunostained for alpha-smooth muscle actin (alpha-SMA, a myofibroblast marker) were rare (0.69 +/- 0.48%), but in sclerotic valves of animals fed an atherogenic diet, myofibroblasts were spatially clustered and abundant (31.2 +/- 6.3%). In cultured VIC populations from normal valves, SMA-positive myofibroblasts were also spatially clustered, abundant (21% positive cells after 1 passage), and stained for collagen type I and vimentin but not desmin. For an analysis of stem cells, two-color flow cytometry of isolated cells stained with Hoechst 33342 demonstrated that 0.5% of VICs were side population cells; none stained for SMA. Upon culture, sorted side population cells generated approximately 85% SMA-positive cells, indicating that some myofibroblasts originate from a rare population with stem cell characteristics. Plating cells on rigid collagen substrates enabled the formation of myofibroblasts after 5 days in culture, which was completely blocked by culture of cells on compliant collagen substrates. Exogenous tensile force also significantly increased SMA expression in VICs. Isotope-coded affinity tags and mass spectrometry were used to identify differentially expressed proteins in myofibroblast differentiation of VICs. Of the nine proteins that were identified, cofilin expression and phospho-cofilin were strongly increased by conditions favoring myofibroblast differentiation. Knockdown of cofilin with small-interfering RNA inhibited collagen gel contraction and reduced myofibroblast differentiation as assessed by the SMA incorporation into stress fibers. When compared with normal valves, diseased valves showed strong immunostaining for cofilin that colocalized with SMA in clustered cells. We conclude that in VICs, cofilin is a marker for myofibroblasts in vivo and in vitro that arise from a rare population of stem cells and require a rigid matrix for formation.

Differential expression of ionic conductances in interstitial cells of Cajal in the murine gastric antrum

Two distinct populations of interstitial cells of Cajal (ICC) exist within the tunica muscularis of the gastric antrum, and these cells serve different physiological functions. One population of ICC generates and actively propagates electrical slow waves, and the other population of ICC is innervated by excitatory and inhibitory motor neurons and mediates enteric motor neurotransmission. In spite of the key role of ICC in gastric excitability, little is known about the ionic conductances that underlie the functional diversity of these cells. In the present study we isolated ICC from the murine gastric antrum and investigated the Ca(2+)-dependent ionic conductances expressed by these cells using the patch clamp technique. Conductances in ICC were compared with those expressed in smooth muscle cells. The cells studied were identified by RT-PCR using cell-specific primers that included Myh11 (smooth muscle cells), Kit (ICC) and Uchl1 (enteric neurons) following electrophysiolgical recordings. Distinct ionic conductances were observed in Kit-positive cells. One group of ICC expressed a basal non-selective cation conductance (NSCC) that was inhibited by an increase in [Ca(2+)](i) in a calmodulin (CaM)-dependent manner. A second population of ICC generated spontaneous transient inward currents (STICs) and expressed a basal noisy NSCC that was facilitated by an increase in [Ca(2+)](i) in a CaM-dependent manner. The [Ca(2+)](i)-facilitated NSCC in ICC was blocked by the Cl(-) channel antagonists 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), anthracene-9-carboxylate (9-AC) and niflumic acid. These data suggest that distinct NSCC are expressed in subpopulations of ICC and these conductances may underlie the functional differences of these cells within the gastric antrum.

Lung stem cells

The lung is a relatively quiescent tissue comprised of infrequently proliferating epithelial, endothelial, and interstitial cell populations. No classical stem cell hierarchy has yet been described for the maintenance of this essential tissue; however, after injury, a number of lung cell types are able to proliferate and reconstitute the lung epithelium. Differentiated mature epithelial cells and newly recognized local epithelial progenitors residing in specialized niches may participate in this repair process. This review summarizes recent discoveries and controversies, in the field of stem cell biology, that are not only challenging, but also advancing an understanding of lung injury and repair. Evidence supporting a role for the numerous cell types believed to contribute to lung epithelial homeostasis is reviewed, and initial studies employing cell-based therapies for lung disease are presented. As a detailed understanding of stem cell biology, lung development, lineage commitment, and epithelial differentiation emerges, an ability to modulate lung injury and repair is likely to follow.

Altered distribution of interstitial cells in the guinea pig bladder following bladder outlet obstruction

We investigated the effects of bladder outlet obstruction (BOO) on the distribution of interstitial cells (ICs) in the guinea-pig bladder. Bladder overactivity of BOO animals was validated with urodynamic studies. Immunohistochemical analyses for Kit and vimentin as markers for ICs were performed on both BOO and control bladders. Morphological and functional properties of detrusor smooth muscle (DSM) were examined with alpha-smooth muscle actin staining and intracellular recording, respectively. Electron microscopy was also carried out to characterize ultrastructural morphology of ICs. Two weeks after surgery, BOO animals showed an increased voiding frequency and a reduced voiding volume. Filling cystometry demonstrated a frequent incidence of non-voiding contractions, a reduced interval between voiding contractions and an increased voiding pressure in BOO bladders. In BOO bladders, the thickness of suburothelial and subserosal connective tissue layers was increased, whilst that of detrusor smooth muscle (DSM) layer was less affected. Population of Kit or vimentin immunoreactive ICs was increased in subserosal layers, and their distribution was altered in suburotherial layer in BOO bladders. Neither alpha-actin immunoreactivity nor spontaneous electrical activity of DSM was altered in BOO bladders. ICs were characterized by their numerous mitochondria and caveolae, and had a close contact with each other and with neighboring DSM or nerves. These results demonstrated the increased population of ICs in the BOO guinea-pig model for the first time, and suggest that the altered distribution of ICs may contribute to the pathophysiology of bladder overactivity.

Immunohistochemical and ultrastructural characteristics of interstitial cells of Cajal in the rabbit duodenum. Presence of a single cilium

Santiago Ramón y Cajal discovered a new type of cell related to the myenteric plexus and also to the smooth muscle cells of the circular muscle layer of the intestine. Based on their morphology, relationships and staining characteristics, he considered these cells as primitive neurons. One century later, despite major improvements in cell biology, the interstitial cells of Cajal (ICCs) are still controversial for many researchers. The aim of study was to perform an immunohistochemical and ultrastructural characterization of the ICCs in the rabbit duo-denum. We have found interstitial cells that are positive for c-Kit, CD34 and nestin and are also positive for Ki67 protein, tightly associated with somatic cell proliferation. By means of electron microscopy, we describe ICCs around enteric ganglia. They present triangular or spindle forms and a very voluminous nucleus with scarce per-inuclear chromatin surrounded by a thin perinuclear cytoplasm that expands with long cytoplasmic processes. ICC processes penetrate among the smooth muscle cells and couple with the processes of other ICCs located in the connective tissue of the circular muscle layer and establish a three-dimensional network. Intercellular con-tacts by means of gap-like junctions are frequent. ICCs also establish gap-like junctions with smooth muscle cells. We also observe a population of interstitial cells of stellate morphology in the connective tissue that sur-rounds the muscle bundles in the circular muscle layer, usually close to nervous trunks. These cells establish different types of contacts with the muscle cells around them. In addition, the presence of a single cilium show-ing a structure 9 + 0 in an ICC is demonstrated for the first time. In conclusion, we report positive staining c-kit, CD34, nestin and Ki 67. ICCs fulfilled the usual transmission electron microscopy (TEM) criteria. A new ultrastructural characteristic of at least some ICCs is demonstrated: the presence of a single cilium. Some populations of ICCs in the rabbit duodenum present certain immunohistochemical and ultrastructural characteristics that often are present in progenitor cells.

In-Situ Deformation of the Aortic Valve Interstitial Cell Nucleus Under Diastolic Loading

Within the aortic valve (AV) leaflet resides a population of interstitial cells (AVICs), which serve to maintain tissue structural integrity via protein synthesis and enzymatic degradation. AVICs are typically characterized as myofibroblasts, exhibit phenotypic plasticity, and may play an important role in valve pathophysiology. While it is known that AVICs can respond to mechanical stimuli in vitro, the level of in vivo AVIC deformation and its relation to local collagen fiber reorientation during the cardiac cycle remain unknown. In the present study, the deformation of AVICs was investigated using porcine AV glutaraldehyde fixed under 0-90 mm Hg transvalvular pressures. The resulting change in nuclear aspect ratio (NAR) was used as an index of overall cellular strain, and dependencies on spatial location and pressure loading levels quantified. Local collagen fiber alignment in the same valves was also quantified using small angle light scattering. A tissue-level finite element (FE) model of an AVIC embedded in the AV extracellular matrix was also used explore the relation between AV tissue- and cellular-level deformations. Results indicated large, consistent increases in AVIC NAR with transvalvular pressure (e.g., from mean of 1.8 at 0 mm Hg to a mean of 4.8 at 90 mm Hg), as well as pronounced layer specific dependencies. Associated changes in collagen fiber alignment indicated that little AVIC deformation occurs with the large amount of fiber straightening for pressures below approximately 1 mm Hg, followed by substantial increases in AVIC NAR from 4 mm Hg to 90 mm Hg. While the tissue-level FE model was able to capture the qualitative response, it also underpredicted the extent of AVIC deformation. This result suggested that additional micromechanical and fiber-compaction effects occur at high pressure levels. The results of this study form the basis of understanding transvalvular pressure-mediated mechanotransduction within the native AV and first time quantitative data correlating AVIC nuclei deformation with AV tissue microstructure and deformation.

Enteric neuropathology of the terminal ileum in patients with intractable slow-transit constipation

Slow-transit constipation is usually considered a colonic motor disorder. However, there is some evidence that abnormalities may be present in locations other than the colon. In particular, several studies have reported abnormal motor activity of the small bowel in these patients. We evaluated the neuropathological aspects of the terminal ileum in patients with slow-transit constipation to see whether abnormalities are present that may explain an abnormal motility of the small intestine. Specimens of the terminal ileum were obtained from 16 female patients (age range, 42-76 years) with slow-transit constipation undergoing surgery for intractable symptoms. Fifteen age- and sex-matched controls were used for comparison. Histologic and immunohistochemical evaluation of the myenteric plexus and the smooth muscle of the proximal ileal resection margin was carried out by means of hematoxylin and eosin, trichrome and periodic acid-Schiff stain, neuron-specific enolase, S-100, CD117, CD34, anti-alpha-actin, desmin, and vimentin antibodies. The patient group displayed a significantly reduced number of glial cells, compared with controls, in both the submucosal and the myenteric plexus. Only 1 of the 3 populations of interstitial cells of Cajal (that associated with the deep muscular plexus) was decreased in patients. No differences were found between patients and controls concerning ganglia neurons, fibroblast-like cells, enteric neurons, apoptotic phenomena, and smooth muscle. Patients with slow-transit constipation display neuropathological abnormalities of the terminal ileum to a lesser extent than those we previously found in the colon, which might explain the abnormal motor aspects sometimes found in these patients.

Endogenous nitric oxide/cGMP signalling in the guinea pig bladder: evidence for distinct populations of sub-urothelial interstitial cells

We have examined structures that may operate by using nitric oxide (NO)/soluble guanylyl cyclase (sGC) signalling in the lamina propria of the guinea pig bladder. Cells on the luminal surface of the urothelium and sub-urothelial interstitial cells (SU-ICs) responded to NO with a rise in cGMP. The distribution of these different cells varied between the base, lateral wall and dome. In the base, two regions were identified: areas with sparse surface urothelial cells and areas with a complete covering. A layer of cGMP-positive (cGMP(+)) cells (up to 10 cells deep) was found in the base. cGMP(+)/SU-ICs were also observed in the lateral wall. However, here, the cGMP(+) cells were confined to a layer of only 1-2 cells immediately below the basal urothelial layer (basal cGMP(+)/SU-ICs). Below these cGMP(+)/SU-ICs lay cells that had a similar structure but that showed little cGMP accumulation (deep cGMP(-)/SU-ICs). Both basal and deep SU-ICs expressed the beta1 subunit of sGC and the cGMP-dependent protein kinase I (cGKI), suggesting that the deep SU-ICs can sense NO and signal via cGMP. By using BAY 41-2272, a sensor of endogenous NO production, NO-dependent cGMP synthesis was observed primarily in the basal SU-ICs. A third population of cGKI(+)/cGMP(-) cells was seen to lie immediately below the basal urothelial layer. These cells ("necklace" cells) were less numerous than SU-ICs and extended linking processes suggesting a network. The specific functions of these structures are not known but they may contribute to the emerging multiple roles of the urothelium associated with the generation of bladder sensation.

Bone Marrow–Derived Cells Do Not Contribute Significantly to Collagen I Synthesis in a Murine Model of Renal Fibrosis

Interstitial fibroblasts play a central role in kidney fibrosis. Their origin is debated, with recent data indicating a contribution of bone marrow (BM)-derived cells to the expanded population of interstitial cells after kidney damage in animals and humans. This study investigated whether these BM-derived cells would respond appropriately to a fibrotic drive by producing collagen. A transgenic mouse that expresses both luciferase and beta-galactosidase reporter molecules under the control of a 17-kb promoter and enhancer element of the gene encoding the alpha2 chain of the collagen I was used. Male transgenic BM was transplanted into female wild-type C57BL/6 mice (n=14), and unilateral ureteric obstruction was performed later to induce renal fibrosis. In the obstructed kidney of the BM-chimeric female mice, a mean of 8.6% of smooth muscle actin-positive interstitial cells were Y chromosome positive. Increased collagen I mRNA in the obstructed kidney was detected by in situ hybridization. No luciferase activity was detected by enzyme assays in tissue homogenates of BM recipients, and very few luciferase mRNA transcripts were seen, mainly in tubular cells. beta-Galactosidase activity was not a useful reporter molecule because it could not be distinguished from enhanced endogenous beta-galactosidase activity in the obstructed kidney. These results indicate that BM-derived interstitial cells do not make a significant contribution to collagen I synthesis in the context of renal injury.

Male-specific cell migration into the developing gonad is a conserved process involving PDGF signalling

Male-specific migration of cells from the mesonephric kidney into the embryonic gonad is required for testis formation in the mouse. It is unknown, however, whether this process is specific to the mouse embryo or whether it is a fundamental characteristic of testis formation in other vertebrates. The signalling molecule/s underlying the process are also unclear. It has previously been speculated that male-specific cell migration might be limited to mammals. Here, we report that male-specific cell migration is conserved between mammals (mouse) and birds (quail–chicken) and that it involves proper PDGF signalling in both groups. Interspecific co-cultures of embryonic quail mesonephric kidneys together with embryonic chicken gonads showed that quail cells migrated specifically into male chicken gonads at the time of sexual differentiation. The migration process is therefore conserved in birds. Furthermore, this migration involves a conserved signalling pathway/s. When GFP-labelled embryonic mouse mesonephric kidneys were cultured together with embryonic chicken gonads, GFP+ mouse cells migrated specifically into male chicken gonads and not female gonads. The immigrating mouse cells contributed to the interstitial cell population of the developing chicken testis, with most cells expressing the endothelial cell marker, PECAM. The signalling molecule/s released from the embryonic male chicken gonad is therefore recognised by both embryonic quail and mouse mesonephric cells. A candidate signalling molecule mediating the male-specific cell migration is PDGF. We found that PDGF-A and PDGF receptor-α are both up-regulated male-specifically in embryonic chicken and mouse gonads. PDGF signalling involves the phosphotidylinositol 3-kinase (PIK3) pathway, an intracellular pathway proposed to be important for mesonephric cell migration in the mammalian gonad. We found that a component of this pathway, PI3KC2α, is expressed male-specifically in developing embryonic chicken gonads at the time of sexual differentiation. Treatment of organ cultures with the selective PDGF receptor signalling inhibitor, AG1296 (tyrphostin), blocked or impaired mesonephric cell migration in both the mammalian and avian systems. Taken together, these studies indicate that a key cellular event in gonadal sex differentiation is conserved among higher vertebrates, that it involves PDGF signalling, and that in mammals is an indirect effect of Sry expression.

Stem Cell Function, Self-Renewal, and Behavioral Heterogeneity of Cells from the Adult Muscle Satellite Cell Niche

Satellite cells are situated beneath the basal lamina that surrounds each myofiber and function as myogenic precursors for muscle growth and repair. The source of satellite cell renewal is controversial and has been suggested to be a separate circulating or interstitial stem cell population. Here, we transplant single intact myofibers into radiation-ablated muscles and demonstrate that satellite cells are self-sufficient as a source of regeneration. As few as seven satellite cells associated with one transplanted myofiber can generate over 100 new myofibers containing thousands of myonuclei. Moreover, the transplanted satellite cells vigorously self-renew, expanding in number and repopulating the host muscle with new satellite cells. Following experimental injury, these cells proliferate extensively and regenerate large compact clusters of myofibers. Thus, within a normally stable tissue, the satellite cell exhibits archetypal stem cell properties and is competent to form the basal origin of adult muscle regeneration.

Cyclic GMP-gated channels are co-localized with a population of Interstitial cells, but not with neuronal NOS, in the rat urethra

The present study further analyse the presence of cyclic nucleotide-gated (CNG) channels and its involvement in the nitrergic urethral relaxation. Our functional results confirm and extend to the rat those previously found in the sheep urethra [1]. Moreover, rat urethra is even more sensitive to the effect of L-cis-diltiazem, a selective inhibitor of CNG channels. Double-labelling immunofluorescence was performed on fixed sections (30 μm) of rat urethra to visualize CNG1 subtype channels (goat anti-rat CNG1//FITC-conjugated donkey anti-goat) and its colocalization with either neuronal NOS(rabbit anti-rat nNOS//Rhodamine-conjugated donkey anti-rabbit) or vimentin(mouse anti-vimentin//Rhodamine-conjugated donkey anti-mouse) positive cells. Micrographs of the from 2nd International Conference of cGMP Generators, Effectors and Therapeutic Implications Potsdam, Germany, 10–12 June, 2005

The effects of cellular contraction on aortic valve leaflet flexural stiffness

The aortic valve (AV) leaflet contains a heterogeneous interstitial cell population composed predominantly of myofibroblasts, which contain both fibroblast and smooth muscle cell characteristics. The focus of the present study was to examine aortic valve interstitial cell (AVIC) contractile behavior within the intact leaflet tissue. Circumferential strips of porcine AV leaflets were mechanically tested under flexure, with the AVIC maintained in the normal, contracted, and contraction-inhibited states. Leaflets were flexed both with (WC) and against (AC) the natural leaflet curvature, both before and after the addition of 90 mM KCl to elicit cellular contraction. In addition, a natural basal tonus was also demonstrated by treating the leaflets with 10 μ M thapsigargin to completely inhibit AVIC contraction. Results revealed a 48% increase in leaflet stiffness with AVIC contraction (from 703 to 1040 kPa, respectively) when bent in the AC direction ( p = 0.004 ) , while the WC direction resulted only in 5% increase (from 491 to 516.5 kPa, respectively—not significant) in leaflet stiffness in the active state. Also, the loss of basal tonus of the AVIC population with thapsigargin treatment resulted in 76% (AC, p = 0.001 ) and 54% (WC, p = 0.036 ) decreases in leaflet stiffness at 5 mM KCl levels, while preventing contraction with the addition of 90 mM KCl as expected. We speculate that the observed layer dependent effects of AVIC contraction are primarily due to varying ECM mechanical properties in the ventricularis and fibrosa layers. Moreover, while we have demonstrated that AVIC contractile ability is a significant contributor to AV leaflet bending stiffness, it most likely serves a role in maintaining AV leaflet tissue homeostasis that has yet to be elucidated.

Identification of persistently altered gene expression in the kidney after functional recovery from ischemic acute renal failure

Recovery from ischemic acute renal failure (ARF) involves a well-described regenerative process; however, recovery from ARF also results in a predisposition to a progressive renal disease that is not well understood. This study sought to identify alterations in renal gene expression in postischemic, recovered animals that might play important roles in this progressive disorder. RNA isolated from sham-operated control rats or rats 35 days after recovery from bilateral ischemia-reperfusion (I/R) injury was compared using a cDNA microarray containing approximately 2,000 known rat genes. A reference hybridization strategy was utilized to define a 99.9% interval and to identify 16 genes that were persistently altered after recovery from I/R injury (12 were upregulated and 4 were downregulated). Real-time PCR verified the altered expression of six of eight genes that had been positively identified. Several genes that were identified had not previously been evaluated within the context of ARF. S100A4, a specific marker of fibroblasts, was identified in a population of interstitial cells that were present postischemic injury. S100A4-positive cells were also identified in tubular cells at earlier time points postischemia. Genes associated with calcification, including osteopontin and matrix Gla protein, were also enhanced postischemic injury. Several proinflammatory genes were identified, including complement C4, were enhanced in postischemic tissues. Conversely, renal kallikrein expression was specifically reduced in the postischemic kidney. In summary, genes with known inflammatory, remodeling, and vasoactive activities were identified in rat kidneys after recovery from ARF, some of which may play a role in altering long-term renal function after recovery from ARF.

CGMP‐generating cells in the bladder wall: identification of distinct networks of interstitial cells

To identify cells which might contribute to the complex physiological responses of the guinea-pig bladder, and specifically to describe the distribution and types of cell in the bladder wall of the guinea pig which respond to nitric oxide (NO) with an increase in intracellular cGMP, i.e. putative interstitial cells (ICs). The whole bladder was removed from 11 male guinea pigs killed by cervical dislocation. Sections of the bladder wall, from the dome lateral wall and base, were isolated and incubated separately in Krebs' solution at 36 degrees C, gassed with 95% O(2) and 5% CO(2), and containing 1 mmol/L of the nonspecific phosphodiesterase inhibitor isobutyl-methyl-xanthene. Individual pieces of tissue were then exposed to 100 micromol/L of the NO donor NONOate for 10 min; control tissues remained in Krebs' solution. Tissues were then fixed in 4% paraformaldehyde and processed for immunohistochemistry. cGMP and neuronal NO synthase (nNOS) were subsequently visualized using appropriate primary and secondary antibodies. Cells responding to NO with an increase in cGMP were detected in the dome, lateral wall and base, with positive cells in the thin outer surface of the wall (muscle coat), associated with muscle bundles in an outer layer of muscle, and in a region immediately beneath the urothelium. These cells (not urothelium, smooth muscle or vascular) are described as interstitial cells. Superficial urothelial umbrella cells were apparent and were strongly cGMP-positive. A high density of interstitial cells was associated with muscle bundles on the outer aspects of the wall, while few cells were detected on inner bundles. Thus there appeared to be two distinct types of muscle, inner and outer, with no obvious orientation of the fibres in each layer. Both muscle groups contained fibres expressing nNOS. In the outer muscle layer most of these fibres co-localized with cGMP, suggesting that different populations of nerves innervate each layer. There were more nNOS-positive fibres in the base of the bladder than in the dome. Three populations of cGMP-positive interstitial cells were associated with the outer muscle layer; cells in the outer surface (muscle coat interstitial cells, MC-ICs), cells on the surface of the bundles (superficial, SM-ICs) and cells within the muscle bundles (intramuscular, IM-ICs). The IM-ICs form a network in close apposition to the smooth muscle cells while the SM-ICs may connect adjacent muscle bundles and connect to the MC-ICs. Thus, there is a network linking potentially the muscle cells in the outer muscle bundles. cGMP-positive cells were also detected in the suburothelial layer (suburothelial, SU-ICs) which had a different structure to the cells associated with muscle, had a oval cell bodies with bifurcating processes and appeared to form a complex network; they were prevalent in the base and virtually absent in the dome. There are structures within the bladder wall that can be identified and categorized by the ability of the constituent cells to increase intracellular cGMP in response to NO; these cells have been defined as ICs. Two distinct networks were identified, one associated with the outer muscle layers and another lying immediately beneath the urothelium, predominantly in the base of the bladder. The functions of these cells and networks are unknown; their possible roles in complex motor activity, urothelial signalling and bladder pathophysiology are discussed.

Histological organization of roe deer testis throughout the seasonal cycle: variable and constant components of tubular and interstitial compartment.

Seasonally regulated breeding in roe deer, Capreolus capreolus, is associated with significant changes in testis mass, structure and function. This study has quantified seasonal changes of morphometric parameters and cellular composition in roe deer testis parenchyma. Tissue samples were collected bimonthly during a complete annual cycle. Morphometric parameters of seminiferous tubules were measured and the number of different cell types was counted using a computer-aided image-analyzing system. A scheme of eight tubular epithelium stages for active spermatogenesis was devised according to the spermatid development. Stage I is characterized by the occurrence of new round spermatids, stage IV by spermiation and stage VIII by the meiotic division of spermatocytes. The average diameter of seminiferous tubules varied between 88.4+/-3.6 micro m (February) and 216.8+/-9.2 micro m (June). Also numbers of spermatogonia, spermatocytes and spermatids per tubule cross-section showed considerable seasonal changes. In December and February the germinative epithelium mainly consists of Sertoli cells and spermatogonia. In February, the first differentiated spermatogonia enter meiosis, and in April even spermatids occasionally occur, which reach their highest numbers during the rut in August. Both the expansion and the proportion of tubular and interstitial compartment change seasonally and result in differing cell densities. Assuming numerically constant populations of Sertoli cells and interstitial cells during the entire year, the hypothetical cell numbers per mm(2) of the tubular and interstitial areas were calculated for the seasonally variable total areas of tissue cross-sections. The concordance of these theoretical values with measured cell densities provided evidence that the total numbers of Sertoli cells, as well as interstitial cells, remain really constant throughout the seasonal cycle. The exact quantification of variable and constant components provides basic data for characterization of cell type and stage-specific processes of spermatogenesis.