Edward J. Macarak

Is Lamina Propria Matrix Responsible for Normal Bladder Compliance

Immunohistochemistry using monoclonal and polyclonal antibodies to extracellular matrix proteins is a highly sensitive tool for the characterization of matrix components. For the first time in the normal and noncompliant human bladder we have used antibodies to collagen types I, III and IV, and elastin to provide morphological correlation with mechanical properties noted clinically. In the normal bladder elastin and collagen types I and III showed intense localization in the lamina propria with modest localization in the detrusor layer. In contrast, lamina propria staining in the noncompliant bladder was essentially unchanged, while there was intense localization within the detrusor layer. Significantly, this intense localization consisted of collagen type III and elastin with little increase in type I. Type IV collagen is associated with basement membranes and individual smooth muscle cells, and shows commensurate increase in specimens with muscle hypertrophy and/or hyperplasia. These observations suggest that in the normal bladder the lamina propria may be a major structural capacitance layer with the smooth muscle covering it. The collagen fibers of the lamina propria may gradually unfold during filling, thus, accounting for normal compliance while in the noncompliant bladder the capacitance layer shifts outward to the infiltrated smooth muscle, thus, preventing the normal expansion of the lamina propria. The smooth muscle infiltration consists of a deposition of collagen type III and elastin with little increase of collagen type I, and it results in a loss of compliance. The pattern of localization would suggest that the smooth muscle is responsible for this accumulation.

Comparative adherence of granulocytes to endothelial monolayers and nylon fiber.

Adherence of granulocytes to tissue culture monolayers of endothelium averaged 26.2 +/- 1.3% SEM, which was similar to their adherence on 50-mg nylon fiber columns (27.7 +/- 3.6%). In contrast, adherence to epithelial cells, fibroblasts, kidney cells, and plastic Petri dishes without monolayers was only 12.4, 9.9, 11.1, and 4.3%, respectively. Cyclic nucleotides and adherence-modifying plasma factors induced changes of adherence to endothelium similar to those in nylon fiber columns. Adherence of granulocytes in whole blood was the same as for purified granulocytes in Hanks balanced salt solution. Exposure of endothelial monolayers to 0.18% trypsin for 10 min reduced subsequent granulocyte adherence to 25.2% of control values. Incubation of trypsin-treated monolayers with nutrient medium for 4 h did not improve adherence, but values returned to normal or above by 24 h, with or without serum proteins present in the nutrient medium. The similarity of granulocyte adherence to nylon fiber and to endothelial monolayers in vitro suggests that results with the nylon fiber assay reflect in vivo granulocyte-endothelium interaction. Furthermore, the endothelial monolayer offers a new model for studying this cell-cell relationship in vitro.

Loss of Elasticity in Dysfunctional Bladders: Urodynamic and Histochemical Correlation

To store adequate volumes of urine at low safe pressures an elastic bladder wall is required. We developed 2 new techniques to measure this ability in our urodynamic laboratory: pressure specific bladder volume, which measures the bladder capacity at a given pressure, and dynamic analysis of bladder compliance. Recently, morphometric and histochemical techniques have been used to determine the relative volume of connective tissue in the bladder wall and to measure the 2 major types (I and III) of collagen within the bladder wall. These methods quantitate 3 parameters of bladder ultrastructure: 1) relative volume of per cent connective tissue, 2) ratio of connective tissue to smooth muscle and 3) ratio of type III to type I collagen. These parameters have been shown to be abnormally elevated in patients with dysfunctional bladders compared to normals. The purpose of the study was to describe the ultrastructural changes that occur in the wall of dysfunctional bladders and to determine the ability of these new urodynamic techniques to detect reliably the clinical effect of these histological changes. The study included 29 consecutive patients with dysfunctional bladders necessitating bladder augmentation. All patients had upper tract changes and/or were incontinent despite treatment with clean intermittent catheterization and pharmacotherapy. Preoperative urodynamic evaluation included measurement of the total bladder capacity, pressure specific bladder volume and dynamic analysis of bladder compliance. Full thickness bladder biopsies were obtained from the dome of the bladders during augmentation. The per cent connective tissue and the ratio of connective tissue to smooth muscle were determined for all patients, and 4 unselected patients from this group had the ratio of type III to type I collagen determined. These histological results were compared to previously established normal values. All 29 patients had a decreased pressure specific bladder volume and dynamic analysis of bladder compliance, whereas 9 had a normal total bladder capacity. The per cent connective tissue was 35.19 +/- 2.84 and ratio of connective tissue to smooth muscle was 0.60 +/- 0.08 compared to normal values of 10.6 +/- 0.020 and 0.131 +/- 0.021, respectively (p < 0.05). Ratio of type III to type I collagen was also significantly elevated in the 4 samples analyzed (30.53 +/- 1.37 versus 24.00 +/- 2.50, p < 0.05). We conclude that poor storage function of poorly compliant bladders is secondary to an alteration in the connective tissue content of the bladder wall. Furthermore, these pathological ultrastructural changes are universally reflected by an abnormally low pressure specific bladder volume and dynamic analysis of bladder compliance. This strong association validates the use of these parameters and suggests that they are urodynamic indicators of a loss of elasticity in bladder wall.

Virus Infection of Endothelial Cells Increases Granulocyte Adherence

Adherence of human granulocytes was measured on endothelial monolayers of human and bovine origin, grown in 35-mm Diam petri dishes and in cluster wells. Adherence to human endothelium in petri dishes using 1.0 ml of whole blood averaged 17.9+/-3.7%, and to bovine endothelium was 20.3+/-3.7%. Cluster wells required only 1/5 the endothelial cells needed for petri dishes, and 0.25 ml of whole blood yielded average adherence of 26.2+/-3.4 to human cells and 28.0+/-3.7 to bovine in the wells. The impact of infection of the endothelium by different viruses on subsequent granulocyte adherence was measured. Polio virus produced an acute lytic infection of human endothelial cells, with associated increased adherence to 185.4% of control 24 h after inoculation. Significantly increased adherence was noted at 6 h, before detectable cytopathic effect. Herpes simplex type I caused a similar rapidly lytic infection of bovine endothelium associated with increased adherence to 213.7% of control 6 h after inoculation. This augmented adherence could be demonstrated when granulocytes were suspended in physiologic saline solution, showing that antibody and complement need not be present. Trypsin treatment of infected monolayers did not prevent the augmentation, and supernate from infected monolayers increased the adherence of polymorphonuclear leukocytes to normal, uninfected monolayers. Chronic, slowly lytic infections, lasting 7 d or more, were induced with adenovirus in human endothelium and with measles virus in bovine cells. Adherence increased as virus was noted in the cell cultures on day 4, several days before cytotoxicity was seen. Thus, chronic viral infection of the endothelium appears possible, and results in increased granulocyte adherence. In naturally occurring disease, such an infection may act synergistically with adherent granulocytes to damage the endothelium, and may represent an in vitro model of vasculitis.

Neurons respond directly to mechanical deformation with pannexin-mediated ATP release and autostimulation of P2X7 receptors.

•  Neurons can be damaged when tissues are stretched or swollen; while astrocytes can contribute to this process, the mechanosensitive response from neurons is unclear. •  We show here that isolated retinal ganglion cell neurons respond to mechanical strain with a rapid, sustained release of the neurotransmitter ATP. •  The conduit for ATP release was through pannexin hemichannels, with probenicid, carbenoxelone and 10panx inhibiting release. •  Once released, this ATP acts back on the neurons to autostimulate lethal P2X7 receptors, as A438079, AZ 10606120 and zinc reduced currents in whole cell patch clamp recordings. •  Blocking release of ATP through pannexin channels, or activation of P2X7 receptors, might be neuroprotective for stretched or swollen neurons. •  Stretch‐dependent release of ATP through neuronal pannexins, combined with the autostimulation of the P2X7 receptors, provides a new pathway by which neuronal activity and health can be altered by mechanical strain independently of glial activity.

Strain measurements in cultured vascular smooth muscle cells subjected to mechanical deformation

Early work in the field of biomechanics employed rigorous application of the principles of mechanics to the study of the macroscopic structural response of tissues to applied loads. Interest in the functional response of tissues to mechanical stimulation has lead researchers to study the biochemical responses of cells to mechanical loading. Characterization of the experimental system (i.e., specimen geometry and boundary conditions) is no less important on the microscopic scale of the cell than it is for macroscopic tissue testing. We outline a method for appropriate characterization of cell deformation in a cell culture model; describe a system for applying a uniform, isotropic strain field to cells in culture; and demonstrate a dependence of cell deformation on morphology and distribution of adhesion sites. Cultured vascular smooth-muscle cells were mechanically deformed by applying an isotropic strain to the compliant substrate to which they were adhered. The state of strain in the cells was determined by measurement of the displacements of fluorescent microspheres attached to the cell surface. The magnitude and orientation of principal strains were found to vary spatially and temporally and to depend on cell morphology. These results show that cell strain can be highly variable and emphasize the need to characterize both the loading conditions and the actual cellular deformation in this type of experimental model.

Bladder smooth muscle cells in culture. I: Identification and characterization

This report documents the growth and culture characteristics of human and fetal bovine bladder smooth muscle cells in vitro. Bladder smooth muscle cell strains have been identified by their spindle shaped morphology, noncontact inhibited growth characteristics and the expression of smooth muscle cell specific alpha-actin. Extracellular matrix protein biosynthesis by these cells in vitro has been characterized by metabolic labeling of proteins with [14C] radiolabeled proline and analysis by SDS gel electrophoresis. These studies demonstrate that bladder smooth muscle cells synthesize predominantly types I and III collagen, and fibronectin. In addition type III collagen exists in both a partially processed (pN alpha 1[III]) form and processed form. Complementary immunohistochemical studies show localization of type I, III, and IV collagens, and fibronectin to bladder smooth muscle cell extracellular matrix. We conclude that both fetal bovine and human smooth muscle bladder cells are capable of secreting the classic components of the surrounding connective tissue.

Mechanosensitive release of adenosine 5′-triphosphate through pannexin channels and mechanosensitive upregulation of pannexin channels in optic nerve head astrocytes: A mechanism for purinergic involvement in chronic strain

As adenosine 5′‐triphosphate (ATP) released from astrocytes can modulate many neural signaling systems, the triggers and pathways for this ATP release are important. Here, the ability of mechanical strain to trigger ATP release through pannexin channels and the effects of sustained strain on pannexin expression were examined in rat optic nerve head astrocytes. Astrocytes released ATP when subjected to 5% of equibiaxial strain or to hypotonic swelling. Although astrocytes expressed mRNA for pannexins 1–3, connexin 43, and VNUT, pharmacological analysis suggested a predominant role for pannexins in mechanosensitive ATP release, with Rho kinase contribution. Astrocytes from panx1−/− mice had reduced baseline and stimulated levels of extracellular ATP, confirming the role for pannexins. Swelling astrocytes triggered a regulatory volume decrease that was inhibited by apyrase or probenecid. The swelling‐induced rise in calcium was inhibited by P2X7 receptor antagonists A438079 and AZ10606120, in addition to apyrase and carbenoxolone. Extended stretch of astrocytes in vitro upregulated expression of panx1 and panx2 mRNA. A similar upregulation was observed in vivo in optic nerve head tissue from the Tg‐MYOCY437H mouse model of chronic glaucoma; genes for panx1, panx2, and panx3 were increased, whereas immunohistochemistry confirmed increased expression of pannexin 1 protein. In summary, astrocytes released ATP in response to mechanical strain, with pannexin 1 the predominant efflux pathway. Sustained strain upregulated pannexins in vitro and in vivo. Together, these findings provide a mechanism by which extracellular ATP remains elevated under chronic mechanical strain, as found in the optic nerve head of patients with glaucoma. GLIA 2014;62:1486–1501

Compression and Tension: Differential Effects on Matrix Accumulation by Periodontal Ligament Fibroblasts In Vitro

Human periodontal ligament fibroblasts were subjected to 10% cyclic equibiaxial tensional and compressive forces in vitro. Media supernatants were analyzed for changes in total protein, extracellular matrix proteins type I collagen and fibronectin, as well as MMP expression by gelatin zymography and Western blot. RNA analyses for changes in collagen, MMP-2, and TIMP-2 were carried out by either Real-time PCR and/or Northern blot. Application of compressional forces resulted in decreases in type I collagen and fibronectin protein, Col1A1 RNA, and increases in total protein, MMP-2 protein (latent and active), and MMP-2 RNA. TIMP-2 RNA was unchanged by compressive forces. In contrast, tensional forces increased total protein, type I collagen, Col1A1 RNA, as well as MMP-2 and TIMP-2 RNA. These studies show that cells can perceive two different forms of mechanical stimuli and respond in a differential manner relative to extracellular matrix synthesis and degradation.

Developmental changes in normal fetal bovine whole bladder physiology.

In the present study, normal and experimentally altered whole fetal bovine bladders have been used in vitro to study developmental changes in compliance and capacity. For the first time, we have attempted to define the relative contributions of the detrusor and mucosal layers to bladder compliance. Fetal bovine bladder compliance increases with fetal development. Elimination of the active component of smooth muscle tension improves compliance and increases capacity more than 60% in younger fetuses but only 35% in older fetuses. Active smooth muscle tension as evaluated by whole bladder cystometry is highest in the youngest fetuses and decreases with fetal age. Surgical removal of the detrusor layer (smooth muscle and connective tissue) also increases compliance and capacity substantially in all fetal bladders. These observations show that both smooth muscle and connective tissue are important in the function of the developing fetal bladder. Changes in both of these bladder wall components probably occur during development and are responsible for the physiologic changes observed.