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Biomedical subjects

J Reinartz

Publications and source records attributed to J Reinartz.

At least 19 recordsLinked to original sources
PubMed1996

Dispase-mediated basal detachment of cultured keratinocytes induces urokinase-type plasminogen activator (uPA) and its receptor (uPA-R, CD87).

Keratinocytes synthesize and secrete urokinase-type plasminogen activator (uPA), which is bound in an autocrine manner to a specific receptor (uPA-R, CD87) at their surface. Plasminogen, which is also bound to membrane binding sites, is readily activated by uPA-R-bound uPA. Thus, plasmin for proteolysis of pericullular glycoproteins is provided. While uPA-R and uPA are at low to undetectable levels in keratinocytes of the normal epidermis, both compounds are upregulated in migrating keratinocytes during reepithelialization of epidermal defects and in affected keratinocytes of various epidermal disorders, including bullous dermatoses. We have hypothesized that the disturbance of cell/matrix interactions--a common feature of these diverse pathological situations--induces uPA/uPA-R. Accordingly, we explored whether the dispase-mediated detachment of cultured keratinocytes, which have formed a multilayered epidermis-like structure in vitro, induced uPA and uPA-R. We found increases in uPA secretion, cell-associated uPA activity, and uPA- and uPA-R-antigen in keratinocytes upon dispase-mediated detachment from their growth substratum. The increase was preceded by an increase in uPA-R- and uPA-specific mRNA, which was not observed when the proteinase inhibitor phosphoramidon was added together with dispase. In conclusion, we present evidence that experimental detachment with dispase provides signals for the concomitant upregulation of uPA-R and uPA. The findings support the hypothesis that cell/matrix interactions may influence the expression of the cell surface-associated PA system in human keratinocytes.

Cell Adhesion↗
PubMed1996

Tumor necrosis factor-alpha-induced apoptosis in a human keratinocyte cell line (HaCaT) is counteracted by transforming growth factor-alpha.

The integrity of the human epidermis is guaranteed by a regulated balance of proliferation, differentiation, and physiologic cell death of its main cellular constituent, the epidermal keratinocyte. Physiologic cell death is known as apoptosis and has been recognized as an active regulatory mechanism, complementary to, but functionally opposite of, proliferation. The regulators of the delicate balance between cell death and proliferation are only partially understood in human keratinocytes. Transforming growth factor-alpha (TGF-alpha) has been identified as a positive regulator of proliferation and growth, while tumor necrosis factor-alpha (TNF-alpha) induces apoptosis. Both mediators are thought to influence epidermal keratinocytes under various physiological and pathophysiological conditions. In the current study we have begun to investigate potential regulatory interactions between these two mediators in the human keratinocyte cell line HaCaT. We have found that, when the HaCaT cells were sensitized by the translation inhibitor cycloheximide, TNF-alpha induced apoptosis, as evidenced by nuclear disintegration, DNA fragmentation ("DNA laddering"), and the appearance of soluble DNA/histone complexes. Moreover, we found that the induction of apoptosis was reduced by preincubation of the cells with TGF-alpha. The protective effect of TGF-alpha was abrogated by translation inhibition, indicating that it depended on de novo protein synthesis. Moreover, the protective effect was not accompanied by a reduced surface expression of TNF receptor molecules. We postulate that TNF-alpha-induced apoptosis in HaCaT cells is counteracted by constitutively produced suppressors of apoptosis, the synthesis of which can be downregulated by inhibition of translation and upregulated by the cytokine TGF-alpha.

Apoptosis↗
PubMed1996

Upregulation of cell-surface-associated plasminogen activation in cultured keratinocytes by interleukin-1 beta and tumor necrosis factor-alpha.

Keratinocytes synthesize and secrete urokinase-type plasminogen activator (uPA) which is bound in an autocrine manner to a specific receptor (uPA-R) at the keratinocyte surface. Plasminogen that is also bound to specific membrane binding sites is readily activated by uPA-R-bound uPA. Thus, plasmin is provided for proteolysis of pericellular glycoproteins. The expression of uPA and the uPA-R is confined to migrating keratinocytes during epidermal wound healing, rather than to keratinocytes of the normal epidermis. The regulatory factors of uPA/uPA-R expression in keratinocytes remained largely elusive. Proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta), are present in epidermal wounds. We have therefore tested IL-1 beta and TNF-alpha for their influence on surface-associated plasminogen activation in a human keratinocyte cell line (HaCaT) as well as in primary cultures of normal human epidermal keratinocytes. Both cytokines induced the secretion of uPA into the culture supernatants and a concomitant increase in uPA activity as well as in uPA and uPA-R antigen at the cell surface. The increase was preceded by an increase in specific mRNA. The induction was accompanied by an accelerated uPA-dependent and plasmin-mediated detachment of HaCaT cells from the culture substratum. Taken together, the proinflammatory cytokines IL-1 beta and TNF-alpha induced a coordinated increase in uPA and uPA-R as well as increased pericellular plasmin-mediated proteolysis in human epidermal keratinocytes. This function might be an element of the molecular cell biological events during epidermal wound healing.

Cell Adhesion↗
PubMed1996

Plasminogen activator inhibitor type-2 (PAI-2) in human keratinocytes regulates pericellular urokinase-type plasminogen activator.

Plasminogen activation is observed in the human epidermis during reepithelialization of epidermal defects and under certain pathological conditions. The activation reaction depends on keratinocyte-associated plasminogen activators (PAs), which convert the ubiquitous proenzyme plasminogen into the active trypsin-like serine proteinase plasmin. The PAs are controlled by PA inhibitors (PAIs), of which two major types are known: PAI-1 and PAI-2. In vitro and in vivo keratinocytes express both PAIs. In the current study, we have addressed the possible function of PAI-2 in regulating extracellular PA activity in cultured normal human epidermal keratinocytes (NHEK), the human keratinocyte cell line (HaCaT), and a Ha-ras transfected HaCaT variant (HaRas). PAI-2 was detected intracellularly in all three cell types. Whereas only the NHEK and the HaCaT cells secreted detectable levels of PAI-2 into the culture medium, all three cell types released urokinase-type PA (uPA) into the supernatants. When comparing HaCaT and HaRas cells, we found that the cell lines secreted comparable levels of uPA antigen, whereas the levels of uPA activity were low in the presence of PAI-2, indicating that PAI-2 serves to regulate uPA activity. This assumption was supported by the findings that PAI-2 formed complexes with secreted uPA and that uPA/PAI-2 complexes were present at the surface of the PAI-2-secreting HaCaT cells but not at the surface of PAI-2 nonsecreting HaRas cells. Finally, PAI-2 was found to counteract the uPA-dependent and plasmin-mediated detachment of cultured HaCaT cells. Taken together, our findings indicate that secreted PAI-2 serves to regulate the activity of extracellular uPA in keratinocytes.

Aprotinin↗
PubMed1996

Interleukin-1 beta upregulates tissue-type plasminogen activator in a keratinocyte cell line (HaCaT).

Human keratinocytes synthesize and secrete tissue-type plasminogen activator (tPA). tPA converts the inactive precursor enzyme plasminogen into the trypsin-like proteinase plasmin. tPA is not found in normal epidermis, but in lesional epidermis from patients with a variety of cutaneous diseases, including psoriasis, pemphigus and pemphigoid. The presence of tPA is probably a reaction to the disease process rather than the initiating event in these etiologically and histopathologically diverse lesions. However, the factor(s) that upregulate tPA expression and secretion in keratinocytes have remained largely elusive. We sought to determine whether the inflammatory cytokine interleukin-1 beta (IL-1 beta), which is commonly present in diverse epidermal lesions, influences tPA production. Accordingly, we studied the influence of IL-1 beta on secretion of tPA by cells of the human keratinocyte cell line HaCaT. We found that IL-1 beta increased tPA secretion in these cells. Given the observation that IL-1 beta is a common proinflammatory mediator in cutaneous diseases, our findings may explain the increase in tPA in clinically and etiologically diverse inflammatory epidermal lesions.

Cell Division↗
PubMed1995

Complement component C7 is a plasminogen-binding protein.

Ab deposition, whether by reaction with the specific Ag or by preformed immune complexes, is followed by activation and deposition of complement components. Tissue destruction is observed in the Ab- and complement-induced lesions. The proteolytic enzyme plasmin is thought to participate in the Ab- and complement-mediated organ pathology. Plasmin is generated from plasma-derived plasminogen by cell-derived plasminogen activators (PAs). Two types of PAs are known, urokinase-type PA (uPA) and tissue-type PA (tPA). We investigated whether the PA system and the complement system can interact to promote local plasmin generation. Among the terminal complement components C5b6, C7, C8, and C9, the nonenzymatic component C7 is a plasminogen-binding protein. Radioligand binding studies revealed that the isolated component, as well as C7 after its incorporation into the terminal complement complex C5b-9, can bind plasminogen. Binding was inhibited by the lysine analogues 6-aminohexanoic acid and tranexamic acid, implicating the lysine binding sites of plasminogen into the binding interaction. tPA-mediated plasminogen activation was enhanced in the presence of C7. Based on these findings, an interaction is proposed between the complement system and the plasminogen activator system; a mechanism that may focus plasmin activity to structures that have been tagged by Ab and complement deposition.

Autoradiography↗
PubMed1995

Plasmin abrogates alpha v beta 5-mediated adhesion of a human keratinocyte cell line (HaCaT) to vitronectin.

At cellular surfaces, urokinase-type plasminogen activator (uPA) is bound to a specific receptor (uPA-R). When bound to this receptor, uPA activates plasminogen, which is derived from plasma or the interstitial fluids. Thus, plasmin is provided for proteolysis of pericellular proteinaceous substrates. Here we demonstrate by immunocytology and laser scan microscopy that in the human keratinocyte cell line HaCaT uPA-R and uPA are localized together with the integrin alpha v beta 5 in focal contacts. Via the integrin alpha v beta 5, HaCaT cells adhere to vitronectin in a RGD-dependent manner. Plasmin interfered with the alpha v beta 5-mediated keratinocyte adhesion to vitronectin, most likely via cleavage of vitronectin and destruction of its cell binding function. Our findings demonstrate that plasmin, when generated by the uPA-dependent cell surface-associated pathway of plasminogen activation, can abrogate the cell-binding function of vitronectin and can thus disturb the adhesive interaction with this matrix molecule. In focal contacts molecules are assembled that are crucial for adhesion to vitronectin (i.e., the integrin alpha v beta 5), as well as for the generation of plasmin (i.e., uPA-R and uPA), which can negatively influence the binding interaction. We suggest that the plasmin-mediated abrogation of the interaction between the integrin alpha v beta 5 and vitronectin is a pathway of negative regulation; the codistribution of uPA-R/uPA and alpha v beta 5 in focal contacts may restrict this process to areas of cell/matrix contact.

Amino Acid Sequence↗
PubMed1995

tPA of human keratinocytes: contribution to cell surface-associated plasminogen activation and upregulation by retinoic acid.

We tested distinct variants of a human keratinocyte line (HaCaT) for the expression of tissue-type plasminogen activator (tPA)-specific mRNA, as well as cell surface-associated and secreted tPA. Cells of early passages (passage no. 22) only expressed urokinase plasminogen activator (uPA)- but not tPA-specific mRNA. Cells after prolonged culture (passage no. 44) expressed uPA- and tPA-specific mRNA, but did not release tPA in the extracellular space and did not display surface-associated tPA. HaCaT cells transformed with the c-Ha-ras oncogene (HaCaTras) showed both secreted and surface-associated tPA antigen. The secreted and the surface-associated plasminogen activator (PA)-activity of HaCaTras cells were in part inhibitable by anticatalytic anti-tPA antibodies, thus indicating that tPA contributes to extracellular and surface-associated plasminogen activation. Finally, we demonstrate that tPA secretion of HaCaT 44 cells can be induced by retinoic acid, most likely via interaction of retinoic acid with nuclear-associated retinoic acid-receptor(s).

Base Sequence↗
PubMed1995

Cervical vertebral strain measurements under axial and eccentric loading.

The mid to lower cervical spine is a common site for compression related injury. In the present study, we determined the patterns of localized strain distribution in the anterior aspect of the vertebral body and in the lateral masses of lower cervical three-segment units. Miniature strain gages were mounted to human cadaveric vertebrae. Each preparation was line-loaded using a knife-edge oriented in the coronal plane that was moved incrementally from anterior to posterior to induce compression-flexion or compression-extension loading. Uniform compressive loading and failure runs were also conducted. Failure tests indicated strain shifting to "restabilize" the preparation after failure of a component. Under these various compressive loading vectors, the location which resulted in the least amount of deformation for a given force application (i.e., stiffest axis) was quantified to be in the region between 0.5- 1.0 cm anterior to the posterior longitudinal ligament. The location in which line-loading produced no rotation (i.e., balance point) was in this region; it was also close to where the vertebral body strains change from compressive to tensile. Strain values from line loading in this region produced similar strains as recorded under uniform compressive loading, and this was also the region of minimum strain. The region of minimum strain was also more pronounced under higher magnitudes of loading, suggesting that as the maximum load carrying capacity is reached the stiffest axis becomes more well defined.

Aged↗
PubMed1995

Plasminogen activation by human keratinocytes: molecular pathways and cell-biological consequences.

Keratinocytes are the major cellular constituent of stratified epithelia. Defects in these epithelia are re-epithelialized by keratinocytes migrating from the edge of the defect into the wound. The cells form a monolayer with subsequent differentiation into a multilayered epithelium. It is thought that plasminogen activation by migrating keratinocytes is an important event during re-epithelialization. In the present report we summarize the studies on plasminogen activation by human keratinocytes in vitro and in vivo. Under the aspect of pericellular proteolysis the discussion is focused on the molecular mechanisms of plasminogen activation at the keratinocyte surface and on the cell-biological consequences of pericellular plasmin formation. We describe a cell surface-associated pathway of plasminogen activation which crucially depends on cell surface receptors for (pro)-uPA and plasmin(ogen). uPA bound to its receptor converts cell-bound plasminogen into the active protease plasmin. Compared to plasminogen activation in solution, activation at the keratinocyte cell surface is accelerated by a factor of approx. 7-10, and the plasmin generated and bound at the cell surface is protected against its specific inhibitor alpha 2-antiplasmin. Plasmin thus provided in the pericellular space leads to detachment of cultured keratinocytes from the growth substratum. Plasmin interferes with the adhesion of keratinocytes to fibrin, but not with the adhesion to collagen type I. By demonstrating that keratinocytes of the epithelial outgrowth in healing skin wounds express uPA and the uPA-R and that plasmin(ogen) is colocalized with uPA and/or uPA-R, indirect evidence is provided that this pathway may be operative in vivo. In view of previous findings that plasminogen activation is also observed under certain pathologic conditions in the epidermis, we conclude that plasminogen activation by keratinocytes is rather related to tissue damage and subsequent repair mechanisms than to a specific pathologic situation.

Fibrinolysin↗
PubMed1994

Cyclic compression-flexion loading of the human lumbar spine.

STUDY DESIGN: The present study was designed to investigate the biomechanical behavior of the lumbar spine under controlled complex physiologic situations with chronic input. OBJECTIVE: The objective was to determine the response of the human cadaver lumbar spinal column under repetitive compression-flexion forces. SUMMARY OF BACKGROUND DATA: Studies have been conducted in the past to determine the biomechanical response of the spine under uniaxial or pure forces. There is no methodology that can be used to apply and continuously quantify the fatigue response of the lumbar spinal column under controlled combined complex loading vectors (e.g., compression flexion). METHODS: Intact cadaver lumbar columns (L1-L5) were mounted with the superior end in contact with a ball-transfer mount, inducing a flexion load to the spine while allowing multiple degrees of freedom. The distal portion of the specimen was attached to a six-axis load cell to quantify the force sustained by the specimen during the entire loading cycle. The applied load and piston deformation and the generalized six-axis force histories were gathered as a function of time using a digital data acquisition system. RESULTS: The stiffness versus number of cycles (K-N) response exhibited nonlinear characteristics. The stiffness increased initially and then stabilized after 1,000-2,000 cycles of loading, delineating the viscoelastic characteristics of the spine. The initial stiffness increase before stabilization was found to be significantly different (P < 0.025) compared to the stiffness beyond 2,000 cycles. CONCLUSIONS: The data suggest that the fatigue response can be understood by cyclically loading the ligamentous lumbar spine preparation to approximately 2,000 cycles.

Aged↗
PubMed1994

Correlation of microtrauma in the lumbar spine with intraosseous pressures.

This study was conducted to determine the relationship between intraosseous pressure and vertebral microtrauma in the lumbar spine. Functional spinal units were excised from human cadavers. Radio-opaque dye was injected into the nucleus. Miniature transducers were inserted into the vertebrae to record intraosseous pressures. Compressive loading was applied quasistatically (2 mm/sec) until injury occurred. Movement of the contrast medium was monitored under fluoroscopy. The subchondral endplate was the most vulnerable component for initiation of injury to the lumbar spine segment. In the initial stages of loading, the vertebral endplates gradually bulged outward, with the contrast medium staying within the nucleus. However, at higher physiologic load levels, before reaching the limiting load, the deformations increased, resulting in buckling of one of the endplates. This was followed by the contrast medium impregnating the spongiosa. Microlevel trauma was not observed radiographically after load removal, indicating that one cannot always equate a normal radiograph with normal spinal anatomy. Mean forces, deformations, stiffnesses, energies, and strains were 7.8 kN (+/- 1.4), 5.23 mm (+/- 0.78), 1940 N/mm (+/- 226), 18.7 J (+/- 4.4), and 35.5% (+/- 3.7), respectively. Pressure in the vertebral body containing the injured endplate before the onset of microtrauma was different (P < 0.05) from the pressure after injury; the pressures in the body containing the intact endplate, however, were not statistically different. Significant differences (P < 0.05) in the intraosseous pressures occurred between the two spinal levels at low-level physiologic loads before the onset of microtrauma.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

The receptor for urokinase-type plasminogen activator of a human keratinocyte line (HaCaT).

It is assumed that plasmin participates in pericellular proteolysis in the epidermis. Plasmin is generated by keratinocyte-associated plasminogen activators from the proenzyme plasminogen; plasminogen activation can proceed at the keratinocyte surface. The resultant plasmin interferes with cell to matrix adhesion and does possibly contribute to keratinocyte migration during reepithelialization. Here we describe the receptor for urokinase-type plasminogen activator (uPA-R) in the human keratinocyte cell line HaCaT, which serves to direct plasminogen activation to the cell surface; we relate the receptor to the uPA-R previously described in human myelo-/monocytes. Binding of uPA to the receptor accelerated plasminogen activation by a factor of approximately 10, compared to uPA in solution. Receptor-bound uPA was susceptible to inhibition by the plasminogen activator inhibitors 1 and 2. uPA and uPA-R antigen, as well as uPA activity, were localized to the leading front of expanding sheets of HaCaT cells. Exposure of HaCaT cells to plasminogen was followed by detachment of the cells. Detachment was prevented by an anticatalytic anti-uPA antibody, by the plasmin-specific inhibitor aprotinin, and by the lysine analogue tranexamic acid, the latter of which prevents plasmin(ogen) binding to the cell surface. Our findings support the hypothesis that uPA-mediated plasminogen activation is characteristic of mobile rather than sessile keratinocytes. Moreover, the uPA-R seems to focalize plasminogen activation to the surface of cells at the site of keratinocyte migration.

Base Sequence↗
PubMed1994

Comparison of nuclear magnetic resonance spectroscopy with dual-photon absorptiometry and dual-energy X-ray absorptiometry in the measurement of thoracic vertebral bone mineral density: compressive force versus bone mineral.

31P nuclear magnetic resonance spectroscopy (NMRS) measurements were made on human T2 and T3 vertebral bodies. The bone mineral content (BMC) of isolated vertebral bodies minus the posterior elements and disks was measured using (1) NMRS on a 3.5 T, 85 mm bore GE Medical Systems NT-150 superconducting spectrometer, (2) a Lunar Corporation DPX-L dual-energy X-ray absorptiometry (DXA) scanner in an anterior-posterior (AP) orientation, (3) a Norland Corporation XR26 DXA scanner, also in an AP direction, and (4) a Norland Corporation model 2600 dual-photon absorptiometry (DPA) densitometer in both the AP and superior-inferior (SI) directions. Vertebral body volumes were measured using a water displacement technique to determine volume bone mineral densities (VBMD). They were then compressed to failure using an electrohydraulic testing device, followed by ashing in a muffle furnace at 700 degrees C for 18 h. Correlations of BMC between NMRS and DPA, DXA and ashing were excellent (0.96 < or = r < or = 0.99); in a one-way analysis of variance (ANOVA) test, means were not statistically different at a p level of 0.757. The correlations of VBMD between NMRS and the other methods were not as good (0.83 < or = r < or = 0.95); in a one-way ANOVA test, means were not statistically different at a p level of 0.089. BMC was a better predictor of ultimate compressive failure than VBMD for all six methods. For NMRS, the regression coefficient for BMC was r2 = 0.806, compared with r2 = 0.505 for VBMD. NMRS may prove an alternative to present methods of determining bone mineral.

Absorptiometry, Photon↗
PubMed1994

Failure of synthes anterior cervical fixation device by fracture of Morscher screws: a biomechanical study.

Anterior cervical fixation using the Synthes system has become increasingly popular. Two screw types for anchoring the plates include a "solid" titanium expansion screw and a plasma-sprayed fenestrated expansion screw that permits bony ingrowth. These screws were compared clinically and in the laboratory. In our first 20 cases using Synthes plates secured by Morscher fenestrated screws, 3 failures were observed, unilaterally in 1 patient and bilaterally in 2 others. In the unilateral screw failure, the contralateral screw was "solid" and did not fail. In the mechanical studies, screws were secured in the Synthes plate and embedded into methylmethacrylate and subjected to a sinusoidal bending moment to the mid-shaft of the screw. Load deflection data and cycles to failure were recorded. Fenestrated screws were found to demonstrate nearly twice as much deformation at failure and tolerated significantly fewer cycles to failure than did "solid" screws (p < 0.05). Because benefits of bony ingrowth into the screw are not well identified, the risks of fenestrated screw failure should preclude their routine use.

Adult↗
PubMed1994

Intravertebral pressure changes caused by spinal microtrauma.

Clinical studies indicate variations in intravertebral pressures in patients with and without low back pain. It is known that not all patients with back pain have abnormal lumbar radiographs and, furthermore, microfractures of the endplate may be one of the causes in the origin of low back pain. Consequently, this study was conducted to determine the interrelationship between microtrauma and intraosseous pressures in the lumbar spine. Miniature pressure transducers were inserted into the vertebral bodies and spinous processes of human cadaver spinal units. Radio-opaque medium was injected into the nucleus to fluoroscopically monitor the movement of the fluid from the disc as the preparation was loaded up to the initiation of microtrauma (before reaching the ultimate load-carrying capacity). The onset of injury was evidenced by the microfracture of one of the two endplates and impregnation of the contrast medium into the spongiosa. After relaxation, another cycle of loading was applied by limiting the deflections to the maximum compression sustained under the intact configuration. The load, stiffness, and energy-absorbing capacities were lower (P < 0.05) for the injured specimen compared with the intact configuration. The intraosseous pressures were higher (P < 0.05) in the vertebral body and the spinous process of the vertebra where the endplate exhibited microtrauma in the injured cycle compared with the intact cycle. In contrast, the intraosseous pressures in the vertebral body and the spinous process at the level where the endplate remained intact were not significantly different between the two cycles of loading.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

[The autoimmune dermatosis bullous pemphigoid: deposition and activation of plasminogen in affected epidermis].

Autoimmune dermatoses are associated with antibody deposition, complement activation and subsequent tissue destruction. Plasmin-mediated proteolysis is thought to be one element of tissue destruction in autoimmune bullous dermatoses such as bullous pemphigoid. Biochemical and immunological studies on bullous pemphigoid skin blister fluid disclosed plasmin/alpha 2-antiplasmin and putative plasmin/alpha 2-macroglobulin complexes. Since the formation of plasmin/inhibitor complexes requires active plasmin, our findings indicate previous activation of plasminogen to plasmin in skin lesions.

Enzyme Activation↗