The matrix form of collagen and basal microporosity influence basal lamina deposition and laminin synthesis/secretion by stratified human keratinocytes in vitro

Summary The ability of the collagen matrix form to support the formation of a basal lamina by cultured normal human epidermal keratinocytes (NHEK) was determined using transmission electron microscopy. The collagen matrix forms tested in this study were a) a dry type I collagen film and b) a type I collagen gel. NHEK were grown for 14 days on the following five different substrates: plain plastic culture dishes without the addition of collagen (PP); plain plastic culture dishes overlaid with a dry, aldehyde-crosslinked type I collagen film (DCF-P); plain plastic culture dishes overlaid with an aldehyde-crosslinked type I collagen gel (GEL-P); Millipore Millicell CM microporous membranes overlaid with a dry, aldehyde-crosslinked type I collagen film (DCF-CM); and Millipore Millicell CM microporous membranes overlaid with an aldehyde-crosslinked type I collagen gel (GEL-CM). NHEK maintained for 2 wk on PP and DCF-P were unable to secrete a basal lamina. NHEK grown for 2 wk on the GEL-P and GEL-CM substrates, however, secreted a contiguous basal lamina at the GEL-NHEK interface. To determine if the appearance of this basal lamina correlated with laminin synthesis, laminin was immunoprecipitated from cellular extracts, as well as media from the apical and basal chambers. NHEK grown on the GEL-P substrate synthesized more laminin than did NHEK grown on the other four alternative substrates. In addition, NHEK grown on GEL-CM were able to direct more laminin to the basal compartment than NHEK grown on DCF-CM substrates. Taken together, the data indicate that the matrix form of collagen can influence basal lamina deposition, laminin synthesis, and laminin trafficking in NHEK.     

Composition in situ and in vitro of vascular smooth muscle laminin in the rat

Vascular smooth muscle cells are surrounded by a basal lamina containing an array of macromolecules: included among these are the laminins, a family of oligomeric glycoproteins composed of subunits encoded by different genes. In this study, we have used monoclonal antibodies to several of these subunits, including S-laminin, laminin B2, and laminin B1, to study these proteins in tail artery, superior mesenteric artery, and aorta of rats. In situ, immunostaining for the B2 and S chains was present in the basal lamina, between the smooth muscle cells, throughout the tunica media. In contrast, B1 chain immunostaining was concentrated around cells in the inner media. To investigate whether smooth muscle cells can produce S-laminin, laminin B2, and laminin B1, smooth muscle cells from the superior mesenteric artery were grown in culture and laminin subunit expression determined. In early culture (4 days), immunostaining showed abundant laminin B2 and less B1 synthesis and incorporation into the matrix. Staining for S-laminin was even less intense than for B1 and was localized to areas where cells were densely packed. The same pattern of S-laminin immunostaining was seen during early culture in cells grown on fibronectin, type IV collagen, or gelatin. Immunoblotting detected S-laminin in the conditioned medium from early cultured cells. In later culture (12 days), S-laminin incorporation into the matrix increased markedly compared to incorporation at 4 days. At this time, cells are much more densely packed and multilayered with extensive matrix accumulation. Cyclical stretching of cells in vitro did not increase immunostaining for S-laminin. Together these data show that S-laminin is a component of the arterial media in situ and that in vitro S-laminin is synthesized by smooth muscle cells. Increased incorporation of S-laminin into the matrix in later culture correlates with the presence of a more extensive matrix, suggesting that matrix organization may be critical to S-laminin incorporation.     

Microporosity of the substratum regulates differentiation of MDCK cells in vitro

Summary We have analyzed the ability of the physical substratum to modulate both the ultrastructural and protein synthetic characteristics of the Madin-Darby canine kidney (MDCK) renal cell line. When MDCK cells were seeded on Millipore Millicell CM microporous membrane cell culture inserts they demonstrated a more columnar organization with an increase in cell density sixfold greater than the same cells seeded on conventional plastic substrata. After 1 wk postseeding on the microporous membrane a partial basal lamina was noted, with a contiguous basement membrane being apparent after 2 wk. One-dimensional sodium dodecyl sulfate gel electrophoresis was used to analyze detergent-solubilized proteins from MDCK cells maintained on plastic substrata vs. microporous membranes. When proteins were pulse-labeled with [³⁵S]methionine, a 55 kDa protein was evident in the cytosolic extract of cells grown on collagen, laminin, and nontreated plastic substrata; but this labeled protein was not evident in similar extracts from cells grown on collagen and laminin-coated microporous membranes. To test if the polarized, basement-membrane secreting phenotype of the MDCK cells could be generated on a microporous membrane without pretreatment with any extracellular matrix (ECM) components, cells were seeded on the Millipore Millicell HA (cellulosic) microporous membrane. This type of substrata does not need a coating of ECM components for cell attachment. A partial basement membrane was formed below cells where the basal surface of the cell was planar, but not in areas where the cell formed large cytoplasmic extensions into the filter. This led us to the conclusion that the microporous nature of the substrata can dictate both ultrastructural and protein synthetic activities of MDCK cells. Furthermore, we suggest that both the planar nature of the basal surface and the microporosity of the substrate are corequisites for the deposition of the basement membrane.     

Migrating mesoderm establish a uniform distribution of laminin in the developing grasshopper embryo

The basal lamina is composed of molecules which physically interact to form a network that serves as a migrational scaffold for many cell types. In the developing peripheral nervous system of the grasshopper, neuronal growth cones are intimately associated with the basal lamina as they migrate. Laminin is a major component of the basal lamina and is a potent promoter of neurite outgrowth in vitro. However, it is unclear what the source of laminin is or how the distribution of laminin within the basal lamina is established. To address this question, grasshopper laminin subunit genes were cloned. As expected, laminin was found within the basal lamina throughout the embryo, in particular in the limb bud, where its expression is coincident with the outgrowth and guidance of the Tibial (Til) pioneer neurons. Surprisingly, the synthesis of beta and gamma chains of laminin was restricted to migratory mesodermal cells, while in other nonmigratory tissues, such as epithelium and presumptive muscle, beta and gamma chains of laminin were not detected. In spite of this, laminin immunoreactivity in the basal lamina appears uniform and is available as a substrate for axonal outgrowth.     

Human keratinocytes that have not terminally differentiated synthesize laminin and fibronectin but deposit only fibronectin in the pericellular matrix

Fibronectin and laminin production by human keratinocytes cultured in serum-free, low-calcium medium without a fibroblast feeder layer were examined by several techniques. By indirect immunofluorescence, fibronectin but not laminin appeared as short radial fibrils between the cells and the substratum, and in the pericellular matrix. Synthesis of fibronectin and laminin by 7-day keratinocyte cultures was determined by 18 hr 35S-methionine metabolic labeling followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Fibronectin accounted for 2.9% of total synthesized protein, 26.5% of fluid phase protein secretion, and 4.3% of deposited ECM protein. In contrast, only 0.1% of the total synthesized protein was laminin, little (6.3%) of this product was secreted, and none of this product was deposited in the ECM. Our results indicate that human keratinocytes under culture conditions that prevent terminal differentiation in vitro can synthesize, secrete, and deposit fibronectin in the extracellular matrix. Although these cells synthesize laminin, they secrete very little and deposit no detectable laminin in the matrix under these culture conditions. From these data we believe that fibronectin may play an important role in the interaction of epidermal cells with connective tissue matrix during wound healing or morphogenesis in in vivo situations in which the epidermis is not terminally differentiated.     

Association of laminin with heparan and chondroitin sulfate-bearing proteoglycans in neurite-promoting factor complexes from rat schwannoma cells

The present studies were undertaken to confirm the presence and identity of a putative proteoglycan associated with laminin in neurite-promoting factor complexes isolated from rat schwannoma cell conditioned medium. Sucrose density gradient centrifugation of the complex resolved two laminin-associated Na₂[³⁵S]O₄-labeled peaks which were termed Pools A and B. Both pools had nearly all their [³⁵S] cpms associated with glycosaminoglycan, contained heparan sulfate-proteoglycan core protein antigen and displayed a similarly high neurite promoting potency relative to their laminin contents. However, Pool A contained about twice as many [³⁵S] cpms and twice as much proteoglycan core protein per laminin than Pool B. Seventy percent of Pool A cpms was associated with heparan sulfate and 30% with chondroitin sulfate whereas the inverse was true for Pool B. Treatment with heparitinase and/or chondroitinase ABC caused laminin in either pool to elute at lower salt concentrations from DEAE cellulose. In SDS-PAGE the [³⁵S] cpms of both pools ran with the same mobility as laminin but could be separated from laminin under reducing conditions. The Pool A cpms remained at 900 KD and the Pool B cpms spread over the 200–900 KD range. By rotary shadowing electron microscopy, Pool B fractions contained primarily cross-shaped laminin images, often associated with proteoglycan-like images. Pool A fractions contained i) dense, aggregated images including intact laminin from which emanated proteoglycan-like strands, ii) circular images bearing globular domains and less commonly, iii) distorted cross-shaped laminin-like images. These studies support the existence of at least two forms of laminin-proteoglycan complexes which differ in biochemical, immunochemical and ultrastructural characteristics.Abbreviations HSPG heparan sulfate proteoglycan - ELISA enzyme-linked immunoassay - Pool A Fractions 10–13 in sucrose gradient (Figure 1, lower panel) - Pool B Fractions 14–16 in sucrose gradient (Figure 1, lower panel) - HDPG High density proteoglycan, Fractions 1–3 in cesium chloride gradient, Figure 1, middle panel - CPC cetylpyridinium chloride - GAG glycosaminoglycan Special issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Glycosaminoglycan synthesis by glomeruli in vivo and in vitro

The distribution of glycosaminoglycans in disrupted glomerular fractions was studied using ³⁵SO₄-labeling in vivo and in vitro. The majority of ³⁵S of isolated glomerular basement membrane was found in heparan sulfate after in vivo and in vitro pulses, although the absolute proportion and the degrees of N-sulfation and N-acetylation varied with the conditions of exposure. Varying amounts of chondroitin sulfate and dermatan sulfate were found in the glomerular basement membrane fraction and larger proportions of both of these glycosaminoglycans as well as of heparan sulfate were found in various glomerular fractions. Glomerular glycosaminoglycans distribution studies must take into account the experimental conditions. Basement membrane-like components of the glomerulus such as the mesangial matrix may have varying glycosaminoglycan composition which may be found in association with glomerular basement membrane fractions.     

Retention of differentiated characteristics in human fetal keratinocytes in vitro

Summary Many of the morphologic and biochemical changes that occur during human fetal skin development have been described, yet there has been little experimental analysis of the processes that regulate the development of human fetal skin. This is due in part to difficulties in culturing human fetal epidermal keratinocytes. We have successfully cultured fetal keratinocytes in two different in vitro systems; in a serum-free keratinocyte growth medium (KGM) on tissue culture plastic and cocultured with dermal fibroblasts as spheroidal aggregates. To characterize these fetal keratinocytes in vitro we have assessed their ability to express several markers of epidermal differentiation. Human fetal keratinocytes grown on plastic in KGM stratify and express some of the components of the differentiated epidermis, such as involucrin and the high molecular weight keratins. However, these keratinocytes co-express keratins and vimentin and do not form a structured basement membrane. More characteristics of fetal skin are preserved in mixed aggregates of epidermal keratinocytes and dermal fibroblasts including epidermal stratification, synthesis of basement membrane components, tissue-specific expression of intermediate filaments, involucrin, and expression of high molecular weight keratins. The maintenance of human fetal epidermal keratinocytes in these two in vitro systems and their ability to express many differentiated characteristics suggests that these cultures will be valuable for studies of the molecular mechanisms that regulate the regionally specific differentiation of the human fetal epidermis. This work was supported by the Dermatology Foundation Fellowships funded by Herbert Laboratories and The Upjohn Company and awarded to A. R. H., NIH Training Program in Dermatological Research #5T32AR07472, and NIH grant #5R01HD20996 to A. T. L. Publication no. 74 of the Dermatology Department, University of Rochester, Rochester, NY.     

Antibodies to laminin and immunohistochemical localization of laminin in chronic chagasic cardiomyopathy: a review

Antibodies against laminin were determined by ELISA in forty six patients suffering from Chagas' disease and twenty healthy persons (control group). The patients were divided into three groups according to the severity of clinical, electrocardiographic and echocardiographic studies. Histologic, ultrastructural and immunohistochemical studies were made of endomyocardial biopsy specimens from 10 of these patients with chronic Chagasic cardiomyopathy. Antibodies to laminin were detected in 50% of the patients in each of the three groups. However analysis of the data did not allow us to determine any significant correlation among the severity of the different clinical and non-invasive studies and the level of circulating antibodies to laminin. The highest titers of antilaminin antibodies were detected in the group with severe cardiological alterations (37% of the patients). Histological and electron microscopic observation of myocardial biopsies disclosed marked thickening of the basement membranes of the myocytes, endothelial cells and vascular smooth muscle cells. Light (peroxidase-labeled antibodies) and electron (gold-conjugated antibody) microscopic immunohistochemical methods revealed a positive reaction for laminin in these thickened basement membranes. This thickening may develop as a consequence of: a) an immunologic reaction which is triggered by the presence of a laminin-like molecule on the surfaces of T. cruzi amastigotes and trypomastigotes; b) an immunologic response to direct injury of basement membranes causing some of their components to become antigenic; c) myocardial fibrosis, with synthesis of new connective tissue components, and d) a combination of the preceding factors. The relationship of these changes to antilaminin antibodies remains unclear. From these results, it is not possible to assure a physiopathogenic role for antibodies to laminin in chronic Chagas' cardiomyopathy.     

Differentiation of normal and tumoral human keratinocytes cultured on dermis: Reconstruction of either normal or tumoral architecture

Summary Normal human keratinocytes isolated from skin and squamous carcinoma cells established from a human tumor (TR146 cell line) both exhibit limited morphologic differentiation when they are grown on conventional plastic dishes. However, when they are seeded on human de-epidermized dermis and cultured at the air-liquid interface, they are able to reform an epithelium having the morphology of the tissue of origin (i.e. skin or squamous carcinoma). The distribution in such reconstructed tissues of differentiation markers such as bullous pemphigoid antigen, 67K keratin, involucrin, membrane-bound transglutaminase, and filaggrin was very similar to their distribution in normal skin and squamous carcinoma specimens, respectively. The degree of differentiation is for both cell types extremely sensitive to culture conditions such as retinoic acid concentration, emersion of the cultures, etc. These results show that subcultured normal or tumoral keratinocytes are able to recover their specific morphogenetic potential when cultured in an environment close to their in vivo situation.     

Connective Tissue Growth Factor Is Involved in Structural Retinal Vascular Changes in Long-Term Experimental Diabetes

Early retinal vascular changes in the development of diabetic retinopathy (DR) include capillary basal lamina (BL) thickening, pericyte loss and the development of acellular capillaries. Expression of the CCN (connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed) family member CCN2 or connective tissue growth factor (CTGF), a potent inducer of the expression of BL components, is upregulated early in diabetes. Diabetic mice lacking one functional CTGF allele (CTGF^+/−) do not show this BL thickening. As early events in DR may be interrelated, we hypothesized that CTGF plays a role in the pathological changes of retinal capillaries other than BL thickening. We studied the effects of long-term (6-8 months) streptozotocin-induced diabetes on retinal capillary BL thickness, numbers of pericytes and the development of acellular capillaries in wild type and CTGF^+/− mice. Our results show that an absence of BL thickening of retinal capillaries in long-term diabetic CTGF^+/− mice is associated with reduced pericyte dropout and reduced formation of acellular capillaries. We conclude that CTGF is involved in structural retinal vascular changes in diabetic rodents. Inhibition of CTGF in the eye may therefore be protective against the development of DR.     

Laminin 5 can promote assembly of the lamina densa in the skin equivalent model

Skin equivalents were prepared by culturing human keratinocytes on the surface of type I collagen gel contracted by human skin fibroblasts (dermal equivalents) and by raising the gel to an air-liquid interface. A stratified squamous epithelium was formed with a well-differentiated cornified layer at the top of keratinocyte layers within 7 days after plating of the keratinocytes on the dermal equivalents. Although major basement membrane components such as collagens IV and VII and laminin 5 were detected immunohistochemically at the dermal-epidermal junction, a lamina densa was rarely observed by electron microscopy even in 14-day skin equivalents. When laminin 5 (1, 5 or 20 μg/ml) was added to the culture medium on day 7 through day 14, types IV and VII collagens at the dermal-epidermal junction stained more strongly by immunohistochemistry compared with the control. Patches of lamina densa were present along the epidermal-dermal junction, and vesicles containing electron-opaque sheets approximately 0.6 μm in diameter that reacted with anti-collagen IV antibody were also observed in basal keratinocytes in 14-day skin equivalents by electron microscopy. Morphometric analysis showed that the total length of lamina densa along the dermal-epidermal junction as well as in the vesicles increased up to 180%, 230% or 520% of control cultures by the addition of laminin 5 (1, 5 or 20 μg/ml, respectively). These results suggest that laminin 5 accelerates formation of the lamina densa along dermal-epidermal junction of the skin equivalents, depending on the concentration of laminin 5 supplemented exogenously.     

Connective tissue growth factor is necessary for retinal capillary basal lamina thickening in diabetic mice.

Experimental prevention of basal lamina (BL) thickening of retinal capillaries ameliorates early vascular changes caused by diabetes. Connective tissue growth factor (CTGF) is upregulated early in diabetes in the human retina and is a potent inducer of expression of BL components. We hypothesize that CTGF is causally involved in diabetes-induced BL thickening of retinal capillaries. To test this hypothesis, we compared the effects of streptozotocin (STZ)-induced diabetes on retinal capillary BL thickness between wild-type mice (CTGF+/+) and mice lacking one functional CTGF allele (CTGF+/-). Differences in BL thickness were calculated by quantitative analysis of electron microscopic images of transversally sectioned capillaries in and around the inner nuclear layer of the retina. We show that BL thickening was significant in diabetic CTGF+/+ mice compared with control CTGF+/+ mice, whereas diabetes did not significantly induce BL thickening in CTGF+/- mice. We conclude that CTGF expression is necessary for diabetes-induced BL thickening and suggest that reduction of CTGF levels may be protective against the development of diabetic retinopathy.     

Electric field exposure alters serum melatonin but not pineal melatonin synthesis in male rats

Sprague-Dawley male rats, maintained in a 14:10 h light:dark cycl were exposed for 30 days (starting at 56 days of age) to a 65 kV/m, 60 Hz electric field or to a sham field for 20 h/day beginning at dark onset. Pineal N-acetyltransferase (NAT), hydroxy-indole-o-methyl transferase (HIOMT), and melatonin as well as serum melatonin were assayed. Preliminary data on unexposed animals indicated that samples obtained 4 h into the dark period would reveal either a phase delay or depression in circadian melatonin synthesis and secretion. Exposure to electric fields for 30 days did not alter the expected nighttime increase in pineal NAT, HIOMT, or melatonin. Serum melatonin levels were also increased at night, but the electric field-exposed animals had lower levels than the sham-exposed animals. Concurrent exposure to red light and the electric field or exposure to the electric field at a different time of the day-night period did not reduce melatonin synthesis. These data do not support the hypothesis that chronic electric field exposure reduces pineal melatonin synthesis in young adult male rats. However, serum melatonin levels were reduced by electric field exposure, suggesting the possibility that degradation or tissue uptake of melatonin is stimulated by exposure to electric fields. © 1994 Wiley-Liss, Inc.     

Matrix assembly,regulation, and survival functions of laminin and its receptors in embryonic stem cell differentiation

Laminin-1 is essential for early embryonic basement membrane assembly and differentiation. Several steps can be distinguished, i.e., the expression of laminin and companion matrix components, their accumulation on the cell surface and assembly into basement membrane between endoderm and inner cell mass, and the ensuing differentiation of epiblast. In this study, we used differentiating embryoid bodies derived from mouse embryonic stem cells null for gamma1-laminin, beta1-integrin and alpha/beta-dystroglycan to dissect the contributions of laminin domains and interacting receptors to this process. We found that (a) laminin enables beta1-integrin-null embryoid bodies to assemble basement membrane and achieve epiblast with beta1-integrin enabling expression of the laminin alpha1 subunit; (b) basement membrane assembly and differentiation require laminin polymerization in conjunction with cell anchorage, the latter critically dependent upon a heparin-binding locus within LG module-4; (c) dystroglycan is not uniquely required for basement membrane assembly or initial differentiation; (d) dystroglycan and integrin cooperate to sustain survival of the epiblast and regulate laminin expression; and (e) laminin, acting via beta1-integrin through LG1-3 and requiring polymerization, can regulate dystroglycan expression.     

Asperyellone pretreatment protects HaCaT cells from UVB irradiation induced oxidative damages: Assessment under in vitro and in vivo conditions and at molecular level

The present study explores the UVB protective role of Asperyellone (AY), a secondary metabolite of Aspergillus niger strain AN01. The in vitro UVB protective efficacy of AY was studied using the Human Epidermal keratinocytes cells (HaCaT) cell line. The results suggest the appreciable scavenging of UVB-induced reactive oxygen species in the AY-pretreated cells compared with UVB control. Experimental results on the antioxidant enzymes (Catalase, SOD, LPO, and GPx) profile, histochemical, and molecular analyses support the UVB protective effect of AY in HaCaT cells. Further, the in vivo UVB protective efficacy of AY was studied using animal models and compared with that of commercially available UVB protective agents. Physical, biochemical, and molecular analyses of skin samples emphasized the UVB protective role of AY. Thus, the important beneficial effects of AY have been explored in the present study.     

Demonstration of microfibrils in Bruch's membrane of the eye

The cationic dyes ruthenium red and alcian blue were used to visualize a population of microfibrils in Bruch's membrane, a compound basement membrane located in the uveal tract of the eye between the retinal pigment epithelium and choriocapillaris. Microfibrils were tubular structures, 10-12 nm in diameter, that showed a characteristic beaded pattern. The majority of microfibrils appeared as a dense mantle around the layer of amorphous elastin. Microfibrils and collagen fibers were also present as a loosely organized meshwork in the collagenous zone of the membrane. Microfibrils were also seen along the basal surface of the retinal pigment epithelium where they appeared to insert into the substance of the basal lamina. Ruthenium red staining of microfibrils was not abolished by prior exposure of tissue to several kinds of degradative enzymes. The findings suggest that the elastic properties of Bruch's membrane may depend on both the elastin and microfibrillar components.     

Absence of the basement membrane component nidogen 2, but not of nidogen 1, results in increased lung metastasis in mice

Nidogen 1 and 2 are ubiquitous basement membrane (BM) components. They show a divergent expression pattern in certain adult tissues with a prominent localization of nidogen 2 in blood vessel BMs. Deletion of either nidogen 1 or 2 in mice had no effect on BM formation, suggesting complementary functions. However, studies in these mice revealed isoform-specific functions with nidogen 1-deficient mice showing neurological abnormalities and wound-healing defects not seen in the absence of nidogen 2. To investigate this further nidogen 1- or 2-deficient mice were intravenously injected with B16 murine melanoma cells, and lung metastasis was analyzed. The authors could show that loss of nidogen 2, but not of nidogen 1, significantly promotes lung metastasis of melanoma cells. Histological and ultrastructural analysis of nidogen 1- and 2-deficient lungs did not reveal differences in morphology and ultrastructure of BMs, including vessel BMs. Furthermore, deposition and distribution of the major BM components were indistinguishable between the two mouse strains. Taken together, these results suggest that absence of nidogen 2 might result in subtle changes of endothelial BMs in the lung, which would allow faster passage of tumor cells through these BMs, leading to a higher metastasis rate and more larger tumors.     

Characterization of cationic amino acid transporters (hCATs) 1 and 2 in human skin

In the present study, we characterized the distribution of human cationic amino acid transporters 1 (hCAT1) and 2 (hCAT2) in healthy skin and compared it to psoriatic skin lesions by means of immunohistochemistry. Moreover, we tested the hypothesis that l-arginine and l-ornithine influence the expression and synthesis of hCAT1 and hCAT2 in cell culture experiments by means of real-time-PCR and Western blot. Immunohistochemical comparison between healthy and psoriatic skin revealed a decreased amount of hCAT1, especially in the stratum granulosum of psoriatic skin; the distribution pattern of hCAT2 was not significantly affected in psoriatic skin. Cell culture experiments showed that supraphysiological concentrations of 15 mM l-arginine (72 h) lead to a significant increase of the hCAT1-mRNA and protein expression, whereas other concentrations had no significant influence. In contrast, l-arginine concentrations of 2 mM led to a significant increase of the hCAT2B mRNA-expression after 24 h. However, 48 and 72 h revealed no significant changes and high concentrations (15 mM l-arginine) led to a significant downregulation of the hCAT2B transporter over all time points analyzed. l-ornithine had no effect on the hCAT1 expression of mRNA and protein level. On the other hand the expression of hCAT2B was significantly up regulated at a 5-mM concentration of l-ornithine at all analyzed time points. Other concentrations had no effect. For the first time, the findings yield data about hCAT1 and hCAT2 on protein-level and suggest that l-arginine is a worthwhile object of studies, which investigated l-arginine as a possible therapeutic agent to reduce psoriatic symptoms.