Biomedical subjects

R H Kramer

Publications and source records attributed to R H Kramer.

At least 91 records · Page 5Linked to original sources

PubMed1989

Human microvascular endothelial cells express integrin-related complexes that mediate adhesion to the extracellular matrix.

Microvascular endothelial cells (MEC) must use a set of surface receptors to adhere not only to the vascular basement membrane but, during angiogenic stimulation, to the interstitium. We examined how cultured MEC isolated from human foreskin interact with their subendothelial matrix. MEC were able to attach to diverse extracellular matrix proteins, including fibronectin (Fn), vitronectin (Vn), laminin (Ln), type I and IV collagen, as well as to fibrinogen and gelatin. Adhesion to Fn, but not to laminin or collagens, was specifically blocked in the presence of Arg-Gly-Asp (RGD)-containing peptides. When surface radioiodinated MEC were solubilized and subjected to affinity chromatography on Fn-Sepharose columns, two polypeptides of 150 and 125 kD, corresponding to the integrin heterodimer alpha 5 beta 1, were identified. MEC also express a complex of 150 (alpha) and 95 kD (beta 3) that is related to the Vn receptor. Immunofluorescent staining of MEC cultures with antibodies to the integrin beta 1 subunit demonstrated receptors on the basolateral surface at focal adhesion plaques that co-localized with vinculin and with Fn-positive matrix fibers. Occasionally, antibodies to the Vn receptor stained the vinculin-positive focal adhesion plaques that frequently co-localized with the beta 1 complex. However, in cultures of MEC that were attached to substrates coated with alternating strips of Fn and Vn, the beta 1 complex was preferentially localized to the Fn substrate, while the Vn receptor was concentrated on the Vn substrate. The results indicate that MEC express at least two different heterodimer adhesion receptors that belong to the integrin super-family and appear to have distinct ligand specificities: the Fn receptor and the Vn receptor. These receptors mediate cell adhesion to the extracellular matrix and presumably have an important role in hemostasis and neovascularization.

Binding, Competitive↗

PubMed1989

Solubilization of immobilized protein substrates by metastatic tumor cells.

A new, sensitive automated assay based on the enzyme-linked immunosorbent assay was developed for measuring proteolytic enzyme activity produced by metastatic tumor cells. In this assay, a suitable protein substrate is adsorbed onto the surface of microplates and incubated with dilutions of standard proteinases, viable tumor cells, or tumor-cell-conditioned medium. The loss of immobilized protein due to proteolysis is then detected by means of antibodies directed against the target protein, and is measured by a microplate reader. Both casein and fibronectin were useful as substrates for the assay of various well-defined proteinases. The assay was successfully used to detect degradative activity elaborated by the mouse B16-BL6 melanoma and the human HT1080 fibrosarcoma cell lines. Both cell lines extensively removed immobilized protein substrates when the cells were seeded directly on the protein films. In addition, substrate removal could also be detected in the serum-free culture medium conditioned by the tumor cells. The results indicate that soluble proteinases secreted by tumor cells may be important during tissue invasion.

Animals↗

PubMed1988

Basement-membrane components associated with the extracellular matrix of the lymph node.

Lymph nodes contain an extensive array of extracellular matrix fibers frequently referred to as "reticular fibers" because of their reticular pattern and positive reaction with silver stains. These fibers are known to contain primarily type-III collagen. In the present study, frozen and plastic-embedded sections of mouse and human lymph nodes were subjected to immunostaining with a panel of monospecific antibodies directed against type-IV collagen, type-III collagen, laminin, entactin, and heparan sulfate proteoglycan. Immunofluorescent staining revealed that, in addition to being uniformly stained with antibodies to type-III collagen, these fibers also stained positively with antibodies to type-IV collagen and to other basement-membrane-specific components. Furthermore, the basement-membrane-specific antibodies stained the outer surface of individual fibers. These same type-III collagen-rich fibers were distinct from blood vascular basement membranes since they did not react with antibodies to factor VIII-related antigen, an endothelial-cell-specific marker. The role of these basement-membrane-specific components associated with the reticular fibers of lymphoid tissue is unknown. However, it is possible that the ligands promote attachment of reticular fibroblasts as well as macrophages and lymphocytes to the extracellular matrix fibers.

Animals↗

PubMed1988

Kaposi's sarcoma in AIDS: basement membrane and endothelial cell markers in late-stage lesions.

Early-stage lesions of Kaposi's sarcoma (KS) are composed of single-layered, highly flattened cells lining collagen bundles, whereas late-stage lesions contain densely packed, spindle-shaped cells. We examined the progression of KS lesions in oral mucosa and lymph nodes from patients with AIDS, using antibodies specific for blood vascular endothelial cells (Factor VIII-related antigen) and their basement membrane (Type IV collagen and laminin). In addition, the plant lectin Ulex europaeus, which selectively stains blood vessels, was also used. In early-stage KS lesions, fibronectin, laminin and Type IV collagen were co-distributed at the interface between KS cells and collagen bundles; Factor VIII-related antigen and Ulex europaeus lectin staining was present in vascular channels and in the KS cells. However, in late-stage lesions, few if any KS cells stained with antibody to Factor VIII-associated antigen, although endothelial cells lining blood vessels were positive. Strong staining for laminin and Type IV collagen was present in a pericellular pattern throughout the nodular late-stage lesions. Since lymphatic capillary endothelium does not produce basement-membrane-specific macromolecules, these results support the conclusion that KS cells are related to blood vascular endothelium but eventually lose certain endothelium-specific markers as the cells are transformed into the spindle-shaped cell type.

Acquired Immunodeficiency Syndrome↗

PubMed1988

Modulation of a subthreshold calcium current by the neuropeptide FMRFamide in Aplysia neuron R15.

1. The effect of the endogenous neuropeptide FMRFamide (Phe-Met-Arg-Phe-amide) on the Aplysia bursting pacemaker neuron R15 was studied. Brief local applications of FMRFamide, both on R15 somata in situ, and on R15 somata that were isolated and maintained in primary cell culture, cause a hyperpolarization of the membrane potential and a suppression of spontaneous bursting or beating pacemaker activity. 2. Two-electrode voltage-clamp experiments revealed that FMRFamide decreases the amplitude of an inward current, which activates with depolarization starting at a membrane potential less depolarized than the threshold for action potentials. Previous studies have established that this subthreshold inward current is carried by calcium and is essential for the generation of bursting pacemaker activity in Aplysia neurons. The effect of FMRFamide on the subthreshold inward current of R15 is blocked by divalent cation calcium channel blockers, such as cobalt and manganese, and is unaffected by changing the external concentration of potassium or chloride ions, or addition of blockers of the calcium-activated potassium current, such as external tetraethylammonium or internal EGTA. 3. The subthreshold calcium current of R15 is also decreased by dopamine and by an unidentified synaptic neurotransmitter. These substances mimic and occlude the action of FMRFamide on the subthreshold calcium current, suggesting that all three transmitters converge to affect the same population of calcium channels in neuron R15. 4. The subthreshold calcium current is enhanced by neurotransmitters that elevate cyclic AMP in R15, including serotonin, and the Aplysia neuropeptide egg-laying hormone (ELH). Likewise, the effect of FMRFamide on the subthreshold calcium current is enhanced by serotonin, ELH, and a cyclic AMP analog, suggesting that FMRFamide and cyclic AMP have antagonistic actions on the same population of calcium channels in neuron R15. 5. We conclude that the suppression of spontaneous bursting or beating pacemaker activity in neuron R15 by FMRFamide is due to a decrease in the subthreshold calcium current. The subthreshold calcium current in R15 is a common target for modulation by many different transmitters, acting via several distinct molecular mechanisms.

Animals↗

PubMed1988

An in vitro model to study bacterial invasion of periodontal tissues.

In periodontal disease, the abilities of bacteria to adhere to and degrade in vivo basement membranes should be considered as two of the rate-limiting steps for the potential active or passive invasion of gingival connective tissues. To study these mechanisms in greater detail, we used the PF HR-9 basement-membrane-like matrix to establish an in vitro model of bacterial invasion and degradation. Three gram-negative anaerobic periodontopathic organisms, Bacteroides gingivalis, Fusobacterium nucleatum, and Actinobacillus actinomycetemcomitans, bound in considerably higher numbers to the HR-9 matrix than did 6 strains of gram-positive facultative organisms typically associated with periodontal health. In a further experiment with B. gingivalis, the organism rapidly degraded Type IV collagen, the major macromolecular component constituting the HR-9 matrix. Streptococcus mitis, the nonperiodontopathic bacterium tested, did not degrade this model matrix. This study provides evidence that B. gingivalis, a periodontopathic bacterium, is able to adhere to and degrade basement membranes, whereas nonperiodontopathic organisms appear not to share in these abilities.

Actinobacillus↗

PubMed1988

Mechanism of calcium-dependent inactivation of a potassium current in Aplysia neuron R15: interaction between calcium and cyclic AMP.

In the preceding paper (Kramer and Levitan, 1988), we presented evidence that an inwardly rectifying K+ current (IR) is inactivated by Ca2+ influx accompanying spontaneous bursting activity in the Aplysia neuron R15. In this paper we examine the mechanism that enables Ca2+ to inactivate IR. Since IR is enhanced by cyclic AMP in neuron R15 (Drummond et al., 1980; Benson and Levitan, 1983), we examined the Ca2+-dependent inactivation of IR after application of either serotonin (5-HT), the adenylate cyclase activator forskolin, or a membrane-permeable cAMP analog, all agents that increase cAMP and hence the magnitude of IR. Even though more active IR channels are available under these conditions, less Ca2+-dependent inactivation is observed. This is contrasted with the Ca2+-dependent inactivation of the voltage-gated Ca2+ current (ICa). Elevating cAMP enhances ICa in R15 and also increases its Ca2+-dependent inactivation. Hence the mechanisms whereby Ca2+ inactivates IR and ICa appear to differ from each other. Elevating internal Ca2+ by repeatedly depolarizing the neuron suppresses the response of IR to brief applications of 5-HT, and speeds the relaxation of the response, suggesting that Ca2+ can interfere with the cAMP-dependent activation of IR. One biochemical site where Ca2+ can reduce cellular cAMP is by activating the Ca2+/calmodulin-sensitive form of phosphodiesterase. We have detected such enzyme activity in homogenates of Aplysia abdominal ganglia and extracts of single R15 somata. Inhibitors of the phosphodiesterase activity suppress the Ca2+-dependent inactivation of IR. Finally, we have used a radioimmunoassay to measure cAMP in individual R15 somata, and have found that R15 neurons hyperpolarized for prolonged periods contain more cAMP than do R15 neurons allowed to burst, consistent with the hypothesis that Ca2+ influx reduces cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Calcium-dependent inactivation of a potassium current in the Aplysia neuron R15.

The endogenously bursting pacemaker neuron R15 of Aplysia exhibits an inwardly rectifying K+ current (IR) that was shown previously to be enhanced by various neurotransmitters via the intracellular second messenger, cyclic AMP (Drummond et al., 1980; Benson and Levitan, 1983; Levitan et al., 1987). Here we present evidence that Ca2+ influx, either caused by spontaneous bursting activity or elicited by depolarizing voltage-clamp pulses, causes a large, long-lasting inactivation of IR. The ionic current inactivated by bursts is identified as IR by several criteria: it activates steeply at membrane potentials more negative than the K+ equilibrium potential, has very fast kinetics, is reduced by lowering external K+ from 10 to 2 mM, and is blocked by adding 1 mM Ba2+, 10 mM Cs+, or 5 mM Rb+ to the bathing medium. The peak inactivation of IR is delayed following a single burst of spikes in R15, such that IR decreases maximally by about 20% after 60-90 sec, and then recovers gradually over more than 10 min. The inactivation caused by many bursts of spikes can reduce IR to less than 50% of its initial amplitude. The delay in onset and slow time course of recovery from inactivation of IR suggest that a complex biochemical mechanism underlies the effect of Ca2+ on IR. The effect of depolarization on IR is due specifically to the influx and intracellular accumulation of Ca2+. Depolarizing voltage-clamp pulses are maximally effective at reducing IR when they elicit a large influx of Ca2+, while pulses approaching the Ca2+ equilibrium potential have little effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Measurement of plasminogen activator activity from human fibrosarcoma cells by a new microassay.

Elevated levels of plasminogen activator (PA) activity have been correlated with neoplasia and may have an important role in tumor-cell invasion and metastasis. We have developed a new caseinolytic assay that uses an immunochemical approach to measure the activity of PA elaborated by malignant tumor cells. The highly sensitive assay consists in incubating a source of PA (viable tumor cells, cell extracts, or conditioned medium) with purified plasminogen in microtiter plates precoated with a suitable protein substrate such as casein. Clearance of the immobilized protein substrate by PA-generated plasmin is then measured by a technique based on the enzyme-linked immunosorbent assay. In experiments using urokinase as a source of PA, the assay displayed near linearity over several log units of urokinase activity and could detect as little as 10(-2) Ploug units of PA activity. Besides successfully measuring PA activity produced by the human HT 1080 fibrosarcoma cell line, the assay permitted detection of significant plasminogen-independent proteolytic activity generated by intact tumor cells cultured in direct contact with immobilized protein substrates.

Caseins↗

PubMed1987

Radioanalysis of 82Rb generator eluates.

The activity of 82Rb produced from a 82Sr/82Rb generator is dependent on elution conditions (volume and eluent flow rate) and sampling conditions (time and position of collection). Assays for 82Rb in generator eluates are described using a commercial dose calibrator in a static procedure and a plastic scintillator in a dynamic procedure. Dynamic assays more accurately reflect the 82Rb administered when the eluate is injected directly. Radionuclidic contaminants which may be present with 82Sr are identified and procedures for their measurement are described.

Humans↗

PubMed1987

Augmentation of bursting pacemaker activity by egg-laying hormone in Aplysia neuron R15 is mediated by a cyclic AMP-dependent increase in Ca2+ and K+ currents.

Release of the neuropeptide egg-laying hormone (ELH) from Aplysia bag cell neurons augments the endogenous bursting pacemaker activity of neuron R15. We have studied the ionic mechanisms underlying the effect of ELH in voltage-clamped R15 neurons. Both electrical discharge of the bag cells, which releases endogenous ELH, and application of synthetic ELH on cell R15 result in an increase in two discrete ionic currents. One of these currents activates with hyperpolarization, reverses near the K+ equilibrium potential, is sensitive to the external K+ concentration, and is blocked by addition of 5 mM Rb+ or 1 mM Ba2+ to the bathing medium. This current appears to be identical to the inwardly rectifying K+ current IR. The other current activates with depolarization and is blocked by replacement of external Ca2+ with Co2+ or Mn2+. This current appears to be a voltage-gated Ca2+ current ICa. Both ICa and IR in R15 have previously been shown to be enhanced by the neurotransmitter serotonin, acting via intracellular cyclic AMP. We now report that increasing cyclic AMP in R15, by applying either serotonin or the adenylate cyclase activator forskolin together with a phosphodiesterase inhibitor, mimics and occludes the action of ELH on neuron R15. Furthermore, application of ELH increases the cyclic AMP content of single R15 neurons, as measured by radioimmunoassay. Finally, the effects of ELH are potentiated by a phosphodiesterase inhibitor. These results suggest that ELH augments bursting activity in R15 by causing cyclic AMP-mediated increases in IR and ICa.

Animals↗

PubMed1987

Attachment of oral bacteria to a basement-membrane-like matrix and to purified matrix proteins.

The purpose of this study was to investigate the adherence of oral bacteria to an in vitro basement-membrane-like matrix and to selected individual macromolecular constituents of this matrix. Radiolabeled bacteria were incubated with basement-membrane-like matrices isolated from PF HR-9 cells. Bacteroides gingivalis 33277, Fusobacterium nucleatum FN-2, and Actinobacillus actinomycetemcomitans GA3(A) bound to the matrix in the range of 44 to 70%, considerably higher than the ranges of A. actinomycetemcomitans GA3(NA) and SUNY AB67 (range, 20 to 25%). The attachment of selected strains of gram-positive bacteria such as Streptococcus and Actinomyces spp. was much less frequent (range, 6 to 25%). Competitive inhibition studies demonstrated that preincubating the bacteria with fibronectin significantly decreased the binding of B. gingivalis by 51% but increased the binding of other gram-negative and gram-positive organisms tested. Similarly, preincubating the matrices with antifibronectin antibodies decreased the binding of B. gingivalis by 31%, whereas the other bacteria tested were either unaffected or binding was increased. The adherence of bacteria to purified basement membrane proteins was also investigated. Strain and species differences were seen in binding, but no clear relationship emerged between binding to an intact matrix and binding to isolated matrix proteins. The results of this study suggest that some gram-negative oral bacteria commonly associated with periodontal disease, such as B. gingivalis, A. actinomycetemcomitans, and F. nucleatum, bound in high numbers to basement-membrane-like matrices in vitro. On the other hand, the gram-positive strains tested bound in much fewer numbers. The results suggest that further studies with this in vitro model may aid in understanding the mechanisms by which oral bacteria adhere to basement membranes.

Actinobacillus↗

PubMed1986

Axonal contribution to subthreshold currents in Aplysia bursting pacemaker neurons.

The contribution of axonal activity to the ionic currents which generate bursting pacemaker activity was studied by using the two-electrode voltage-clamp technique in Aplysia bursting neuron somata in conjunction with intraaxonal voltage recordings. Depolarizing voltage-clamp pulses applied to bursting cell somata triggered axonal action potentials. The voltage-clamp current recording exhibited transient inward current "notches" corresponding to each of the axonal spikes. The addition of 50 microM tetrodotoxin (TTX) to the bathing medium blocked the fast axonal spikes and current notches, revealing a slower axonal spike which was blocked by the replacement of external Ca2+ with Co2+. The inward current evoked by applying a depolarizing voltage-clamp pulse in the soma is distorted by the occurrence of the axonal Ca2+ spike. Elimination of the axonal spike, by injecting hyperpolarizing current into the axon, changes both the time course and the magnitude of the inward current. The axonal Ca2+ spikes are followed by a series of Ca2+-dependent afterpotentials: a rapid postspike hyperpolarization, a depolarizing afterpotential (DAP) and, finally, a long-lasting postburst hyperpolarization. The long-lasting hyperpolarization is not blocked by 50 mM external tetraethyl ammonium, an effective blocker of Ca2+-activated K+ current [IK(Ca)], and does not appear to reverse at EK. Hence, the axonal long-lasting hyperpolarization may not be due to IK(Ca). Somatic voltage-clamp pulses in bursting neurons are followed by a slow inward tail current, which is sometimes coincident with a DAP in the axon. In some cells, the amplitude of the slow inward tail current is greatly reduced if axonal spikes and DAPs are prevented by hyperpolarization of the axon, while, in other cells, elimination of axonal activity has little effect. Therefore, the slow inward tail current is not necessarily an artifact of poor voltage-clamp control over the axonal membrane potential but probably results from the activation of an ionic conductance mechanism located partly in the axon and partly in the soma.

Action Potentials↗

PubMed1986

Synthesis of basement membrane-specific macromolecules by cultured human microvascular endothelial cells isolated from skin of diabetic and nondiabetic subjects.

Microvascular endothelial cells isolated from abdominal skin of diabetic and nondiabetic adults were maintained in culture by serial passage. Both cell types showed typical endothelial cell morphology, expressed factor VII-associated antigen, contained Weibel-Palade bodies, and produced an extensive subendothelial extracellular matrix containing type IV (basement membrane) procollagen. Biosynthetic studies using radioactive amino acids indicated that under the conditions of cell culture the matrix proteins newly synthesized by MEC of both cell types were similar in type and amount. Both cell types produced type IV procollagen, laminin, and fibronectin, which were deposited in the matrix. Electron microscopy showed that the matrices of both cell types had a similar multilayered, discontinuous, filamentous ultrastructure. Immunoperoxidase staining showed type IV collagen to be distributed similarly, in a fibrillar meshwork, in both matrices. The extractability of individual matrix macromolecules from both matrices was identical; 4 M urea or guanidine-HCl partially removed fibronectin and thrombospondin, but reducing agent was required to solubilize type IV procollagen. The results suggest that diabetic microangiopathy is not due to an inherent defect in the endothelium, and that this in vitro system may be useful for examining environmental factors possibly involved in its development.

Aged↗

PubMed1986

Invasion of reconstituted basement membrane matrix by metastatic human tumor cells.

A gel-like reconstituted basement membrane matrix containing type IV collagen, laminin, entactin, nidogen, and heparan sulfate proteoglycan was used to examine the invasive properties of human HT1080 fibrosarcoma cells. Within several hours after seeding, the tumor cells initiated a random migration, leaving behind channels etched in the surface of the matrix. Eventually the channels became interconnected into a complex network. As the tumor cells proliferated, the channels became filled until the surrounding matrix was gradually dissolved. Cells then migrated outward, forming the typical disorganized cell monolayer normally observed when fibrosarcoma cells are cultured on plastic surfaces. In contrast to the fibrosarcoma cells, normal skin fibroblasts, while able to attach to the matrix, exhibited minimal migration, tracking, and invasion during the same time period. When tumor cells were seeded onto thick layers of matrix, the cells ultimately invaded downward into the matrix, leaving behind open tunnels. At the front of the invading cells, long irregular pseudopodia projected in the direction of movement. Electron microscopy demonstrated these filopodial and lamellopodial projections to directly extend into the surrounding matrix, with focal clearing of the matrix in the immediate vicinity of these invading pseudopodia. Thus, tumor cell invasion of extracellular matrices, including basal lamina, may proceed by the formation of specialized pseudopodia that not only form adhesion contacts with the matrix but also provide an efficient mechanism for the focal hydrolysis of the matrix at the site of directed cell movement.

Basement Membrane↗

PubMed1985

Type IV collagen synthesis by cultured human microvascular endothelial cells and its deposition into the subendothelial basement membrane.

Cultured microvascular endothelial cells isolated from human dermis were examined for the synthesis of basement membrane specific (type IV) collagen and its deposition in subendothelial matrix. Biosynthetically radiolabeled proteins secreted into the culture medium were analyzed by sodium dodecyl sulfate gel electrophoresis after reduction, revealing a single collagenous component with an approximate Mr of 180 000 that could be resolved into two closely migrating polypeptide chains. Prior to reduction, the 180 000 bands migrated as a high molecular weight complex, indicating the presence of intermolecular disulfide bonding. The 180 000 material was identified as type IV procollagen on the basis of its selective degradation by purified bacterial collagenase, moderate sensitivity to pepsin digestion, immunoprecipitation with antibodies to human type IV collagen, and comigration with type IV procollagen purified from human and murine sources. In the basement membrane like matrix elaborated by the microvascular endothelial cells at their basal surface, type IV procollagen was the predominant constituent. This matrix-associated type IV procollagen was present as a highly cross-linked and insoluble complex that was solubilized only after denaturation and reduction of disulfide bonds. In addition, there was evidence of nonreducible dimers and higher molecular weight aggregates of type IV procollagen. These findings support the suggestion that the presence of intermolecular disulfide bonds and other covalent interactions stabilizes the incorporation of the type IV procollagen into the basement membrane matrix. Cultured microvascular endothelial cells therefore appear to deposit a basal lamina-like structure that is biochemically similar to that formed in vivo, providing a unique model system that should be useful for understanding microvascular basement membrane metabolism, especially as it relates to wound healing, tissue remodeling, and disease processes.

Basement Membrane↗

PubMed1985

Degradation of extracellular matrix by the trophoblastic cells of first-trimester human placentas.

First-trimester human placental villi were cultured on 3H-leucine-labeled extracellular matrices isolated from the PF HR9 and PYS-2 cell lines. Both cell lines produced an extracellular matrix that contained basement membrane-specific macromolecules, including type IV collagen, laminin and proteoglycan. Both matrices promoted outgrowth of cells from the villi which, according to morphological criteria, were identified as cytotrophoblastic cells. As the cells migrated from the attachment site, they caused a marked focal dissolution of the matrix which was accompanied by a concomitant release of 3H-labeled material into the media. Approximately half of this material chromatographed near the inclusion volume of Sephadex G-50, indicating that the labeled matrix components had been degraded. This phenomenon was dependent on the age of the placenta. Second-trimester placental villi also adhered to the matrix, but no areas of dissolution were formed and no significant amounts of radioactivity were released into the medium. These results suggest that culture of first-trimester human placental villi on extracellular matrices may be useful for the study of some of the early embryonic events leading to human implantation, during which the trophoblastic cells erode the uterine epithelium.

Cell Line↗

PubMed1985

Synthesis of extracellular matrix glycoproteins by cultured microvascular endothelial cells isolated from the dermis of neonatal and adult skin.

We examined the synthesis of extracellular matrix macromolecules by human microvascular endothelial cells isolated from the dermis of neonatal (foreskin) and adult (abdominal) skin. Electron microscopy showed that both cell types produced an extracellular matrix that was strictly localized to the subendothelial space. The subendothelial matrices were initially deposited as a single discontinuous layer of filamentous, electron-dense material that progressively became multilayered. Biosynthetic studies indicated that 2-4% of the newly synthesized protein was deposited in the subendothelial matrices by both cell types. Approximately 15-20% of the radiolabeled protein was secreted into the culture medium, and the remainder was confined to the cellular compartment. Biochemical and immunochemical analyses demonstrated the extracellular secretion of type IV collagen, laminin, fibronectin, and thrombospondin by the newborn and adult cells. Whereas type IV collagen was the predominant constituent of the matrix, fibronectin was secreted into the medium, with only small amounts being deposited in the matrix. Thrombospondin was a major constituent of the matrix produced by the newborn foreskin cells but was virtually absent in the matrix elaborated by the adult cells. However, both cell types did release comparable amounts of thrombospondin into their medium. Immunoperoxidase staining for type IV collagen revealed a fibrillar network in the subendothelial matrices produced by both adult and neonatal cells. In contrast, thrombospondin, which was detected only in the matrix of newborn cells, exhibited a spotty and granular staining pattern. The results indicate that the extracellular matrices synthesized by cultured human microvascular endothelial cells isolated from anatomically distinct sites and different stages of development and age are similar in ultrastructure but differ in their macromolecular composition.

Aging↗