CXCR4antagonism as a therapeutic approach to prevent acute kidney injury.

We examined whether antagonism of the CXCR4receptor ameliorates the loss of renal function following ischemia-reperfusion. CXCR4is ubiquitously expressed on leukocytes, known mediators of renal injury, and on bone marrow hematopoietic stem cells (HSCs). Plerixafor (AMD3100, Mozobil) is a small-molecule CXCR4antagonist that mobilizes HSCs into the peripheral blood and also modulates the immune response in in vivo rodent models of asthma and rheumatoid arthritis. Treatment with plerixafor before and after ischemic clamping ameliorated kidney injury in a rat model of bilateral renal ischemia-reperfusion. Serum creatinine and blood urea nitrogen were significantly reduced 24 h after reperfusion, as were tissue injury and cell death. Plerixafor prevented the renal increase in the proinflammatory chemokines CXCL1 and CXCL5 and the cytokine IL-6. Flow cytometry of kidney homogenates confirmed the presence of significantly fewer leukocytes with plerixafor treatment; additionally, myeloperoxidase activity was reduced. AMD3465, a monocyclam analog of plerixafor, was similarly renoprotective. Four weeks postreperfusion, long-term effects included diminished fibrosis, inflammation, and ongoing renal injury. The mechanism by which CXCR4inhibition ameliorates AKI is due to modulation of leukocyte infiltration and expression of proinflammatory chemokines/cytokines, rather than a HSC-mediated effect. The data suggest that CXCR4antagonism with plerixafor may be a potential option to prevent AKI.

Antibody to transforming growth factor-beta ameliorates tubular apoptosis in unilateral ureteral obstruction.

BACKGROUND: Unilateral ureteral obstruction (UUO) is characterized by progressive renal atrophy, renal interstitial fibrosis, an increase in renal transforming growth factor-beta (TGF-beta), and renal tubular apoptosis. The present study was undertaken to determine the effect of a monoclonal antibody to TGF-beta (1D11) in UUO. METHODS: Mechanical stretch was applied to tubular epithelial cells (NRK-52E) by a computer-assisted system. Three doses of 1D11 (either 0.5, 2, or 4 mg/rat) were administered to rats one day prior to UUO and every two days thereafter, and kidneys were harvested at day 13. Fibrosis was assessed by measuring tissue hydroxyproline and mRNA for collagen and fibronectin. Apoptosis was assessed with the terminal deoxy transferase uridine triphosphate nick end-labeling assay. TGF-beta levels were determined by bioassay. Western blot and immunostaining were used to identify proliferating cell nuclear antigen (PCNA), p53, bcl-2, and inducible nitric oxide synthase (iNOS). RESULTS: Stretch significantly induced apoptosis in NRK-52E cells, which was accompanied by an increased release of TGF-beta; 1D11 (10 microg/mL) totally inhibited stretch-induced apoptosis. Control obstructed kidney contained 20-fold higher TGF-beta as compared with its unobstructed kidney; 1D11 neutralized tissue TGF-beta of the obstructed kidney. Control obstructed kidney exhibited significantly more fibrosis and tubular apoptosis than its unobstructed counterpart, which was blunted by 1D11. In contrast, 1D11 significantly increased tubular proliferation. p53 immunostaining was localized to renal tubular nuclei of control obstructed kidney and was diminished by 1D11. In contrast, bcl-2 was up-regulated in the 1D11-treated obstructed kidney. Total NOS activity and iNOS activity of the obstructed kidney were increased by 1D11 treatment. CONCLUSION: The present study strongly suggests that an antibody to TGF-beta is a promising agent to prevent renal tubular fibrosis and apoptosis in UUO.

Renal fibrosis in mice treated with human recombinant transforming growth factor-beta2.

BACKGROUND: The biologic responses to transforming growth factor-beta (TGF-beta) suggest many potential therapeutic applications; however, in the only clinical trial to examine the effect of the systemic administration of a TGF-beta isoform, patients experienced significant but reversible declines in renal function. We studied the effects of administering human recombinant TGF-beta2 to adult mice. METHODS: The effect of daily administration of TGF-beta2 on tissue vasoconstriction, tissue levels of endothelin and angiotensin II, tissue hypoxia, and renal fibrosis were examined. RESULTS: Daily administration of TGF-beta2 at 10 or 100 microg/kg caused apparent tissue vasoconstriction that was visualized by vascular casting, with the largest impact seen in the kidney. Tissue levels of endothelin 1 and angiotensin II were significantly elevated in kidneys of treated mice, as was urinary thromboxane beta2. Renal fibrosis was observed in the cortical tubular interstitium and vasculature, particularly at the cortical-medullary junction and medullary vasa recta; however, glomerular sclerosis was not observed. Fibrosis was correlated to focal tissue hypoxia as determined by immunohistochemical detection of tissue bound pimondazole. CONCLUSION: We conclude that there are significant histopathologic consequences, focused in the kidney, resulting from the daily administration of high doses of human recombinant TGF-beta2, and we propose that selective vascular constriction with consequent tissue hypoxia is a contributing factor.

Nursing education and genetics. Miles to go before we sleep.

The discoveries of the Human Genome Project (HGP), established in 1990 at the National Institutes of Health and the United States Department of Energy, are bringing important new technologies for genetic diagnosis and treatment to nearly all areas of health care delivery. Nurses, who play an integral role in supporting health consumers as they respond to health and illness, require up-to-date genetic knowledge for conducting clinical practice, engaging in nursing research, and educating a new generation of nurses.

Helical (spiral) CT in the evaluation of emergent thoracic aortic syndromes. Traumatic aortic rupture, aortic aneurysm, aortic dissection, intramural hematoma, and penetrating atherosclerotic ulcer.

For the near future, CT will play the critical and dominant role in the evaluation of patients presenting with emergent aortic syndromes. Its convenience, accuracy, and utility in the rapid evaluation of not just the aorta, but the entire thorax, make it ideally suited for use in emergency settings. Further benefits are likely to be realized in speed and resolution with multislice CT, although it is as yet not widely available.

Acute and chronic renal effects of recombinant human TGF-beta2 in the rat.

The expression of transforming growth factor-beta (TGF-beta) correlates with the incidence of renal glomerular and interstitial injury, however, nothing is known of the effect of these proteins on renal hemodynamics. This study examines the renal hemodynamic and morphologic effects of recombinant human TGF-beta2 in normal male Sprague Dawley rats. Acute infusion of TGF-beta (1.2 microg/kg per min) induced no hemodynamic changes, except for a modest though significant fall in mean arterial pressure. Administering TGF-beta2 at varying doses (20, 100, and 400 microg/kg) for 9 wk caused modest increases in systolic BP and proteinuria and minimal tubular interstitial fibrosis, however, renal hemodynamic end points were not significantly altered. TGF-beta2 (800 microg/kg) was also administered to volume-depleted rats for 7 consecutive days. In contrast to the findings in volume-replete animals, administration of TGF-beta2 to volume-depleted rats caused a marked reduction in GFR and medullary blood flow. Histologic fibrosis of the medullary vasa recta and cortical interstitium was seen, but glomeruli were unaffected. Thus, acute and short-term chronic TGF-beta2 administration did not induce major renal changes in the volume-replete state, however, TGF-beta2 combined with volume depletion caused medullary hypoperfusion and reduced GFR.

Solution structures of stromelysin complexed to thiadiazole inhibitors.

Unregulated or overexpressed matrix metalloproteinases (MMPs), including stromelysin, collagenase, and gelatinase. have been implicated in several pathological conditions including arthritis and cancer. Small-molecule MMP inhibitors may have therapeutic value in the treatment of these diseases. In this regard, the solution structures of two stromelysin/ inhibitor complexes have been investigated using 1H, 13C, and 15N NMR spectroscopy. Both-inhibitors are members of a novel class of matrix metalloproteinase inhibitor that contain a thiadiazole group and that interact with stromelysin in a manner distinct from other classes of inhibitors. The inhibitors coordinate the catalytic zinc atom through their exocyclic sulfur atom, with the remainder of the ligand extending into the S1-S3 side of the active site. The binding of inhibitor containing a protonated or fluorinated aromatic ring was investigated using 1H and 19F NMR spectroscopy. The fluorinated ring was found to have a reduced ring-flip rate compared to the protonated version. A strong, coplanar interaction between the fluorinated ring of the inhibitor and the aromatic ring of Tyr155 is proposed to account for the reduced ring-flip rate and for the increase in binding affinity observed for the fluorinated inhibitor compared to the protonated inhibitor. Binding interactions observed for the thiadiazole class of ligands have implications for the design of matrix metalloproteinase inhibitors.

Physiological degradation converts the soluble syndecan-1 ectodomain from an inhibitor to a potent activator of FGF-2.

The activity of fibroblast growth factor 2 (FGF-2) is stringently controlled. Inactive in undisturbed tissues, it is activated during injury and is critical for tissue repair. We find that this control can be imposed by the soluble syndecan-1 ectodomain, a heparan sulfate proteoglycan shed from cell surfaces into wound fluids. The ectodomain potently inhibits heparin-mediated FGF-2 mitogenicity because of the poorly sulfated domains in its heparin sulfate chains. Degradation of these regions by platelet heparanase produces heparin-like heparin sulfate fragments that markedly activate FGF-2 mitogenicity and are found in wound fluids. These results establish a novel physiological control for FGF-2 and suggest new ways to modulate FGF activity.

Identification of a DNA segment exhibiting rearrangement modifying effects upon transgenic delta-deleting elements.

Control of the rearrangement and expression of the T cell receptor alpha and delta chains is critical for determining T cell type. The process of delta deletion is a candidate mechanism for maintaining separation of the alpha and delta loci. Mice harboring a transgenic reporter delta deletion construct show alpha/beta T cell lineage-specific use of the transgenic elements. A 48-basepair segment of DNA, termed HPS1A, when deleted from this reporter construct, loses tight lineage-specific rearrangement control of transgenic elements, with abundant rearrangements of transgenic delta-deleting elements now in gamma/delta T cells. Furthermore, HPS1A augments recombination frequency of extrachromosomal substrates in an in vitro recombination assay. DNA binding proteins recognizing HPS1A have been identified and are restricted to early B and T cells, during the time of active rearrangement of endogenous TCR and immunoglobulin loci. These data are consistent with delta deletion playing an important role in maintaining separate TCR alpha and delta loci.

Human prostate carcinoma cells produce extracellular heparanase.

The degradation of heparan sulfate proteoglycan (HSPG) in basement membranes (BM) has been previously suggested to be accomplished by an endoglycosidase activity called heparanase which has not been isolated outside of platelets. HSPG degradation by heparanase has been associated with tumor cell invasion, angiogenesis, and growth factor function. In this study, we identify heparanase activity biochemically and immunologically in malignant human prostate carcinoma cells (PC-3M), linking platelet heparanase probes with the tumor heparanase activity observed. Concentrated conditioned medium from PC-3M cells was analyzed by a heparin-Sepharose affinity column. Three peaks eluted with 0.15, 0.35, and 0.5 M NaCl. Each peak was analyzed by incubation with 3H-labeled heparin as well as [3H]HSPG from EHS tumor BM. The 0.5 M peak material degraded [3H]-heparin by 17.2%, with little additional degradation by the other peaks in comparison to the conditioned medium from which they were obtained. Likewise, the same amount of the 0.5 M peak accounted for the majority of degradation (30.8%) of 3H-labeled HSPG. Interestingly, for the same amount of 0.5 M peak material, significantly more HSPG was degraded than heparin under the same conditions. In addition, carrageenan-lambda, an inhibitor of glycanase, completely inhibited the degradation of heparin and heparan sulfate proteoglycan by the 0.5 M peak. Using antibody to the N-terminus domain of platelet heparanase, a 60-kDa protein was identified by immunoblot in 0.5 M peak material. Additionally, immunohistochemical staining of human prostate carcinoma specimens showed granular staining at or near the cell membrane and near the luminal surface using antibody to the N-terminus and C-terminus domains of platelet heparanase. In summary, human prostate carcinoma cells show heparanase activity in conditioned medium that degrades heparin and BM HSPG and is detected by antibody to platelet heparanase. In addition, the membrane-associated staining in tissue sections of prostate cancer strongly correlates with the biochemical and immunological detection in conditioned medium of human PC-3M cells.

Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving the alphavbeta3 integrin, osteopontin, and thrombin.

We have identified several mechanisms by which the angiogenic cytokine vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) likely regulates endothelial cells (EC) migration. VPF/VEGF induced dermal microvascular EC expression of mRNAs encoding the alphav and beta3 integrin subunits resulting in increased levels of the alphavbeta3 heterodimer at the cell surface, and VPF/VEGF also induced mRNA encoding osteopontin (OPN), an alphavbeta3 ligand. OPN promoted EC migration in vitro; and VPF/VEGF induction of alphavbeta3 was accompanied by increased EC migration toward OPN. Because thrombin cleavage of OPN results in substantial enhancement of OPN's adhesive properties, and because VPF/VEGF promotes increased microvascular permeability leading to activation of the extrinsic coagulation pathway, we also investigated whether VPF/VEGF facilitates thrombin cleavage of OPN in vivo. Consistent with this hypothesis, co-injection of VPF/VEGF together with OPN resulted in rapid cleavage of OPN by endogenous thrombin. Furthermore, in comparison with native OPN, thrombin-cleaved OPN stimulated a greater rate of EC migration in vitro, which was additive to the increased migration associated with induction of alpha v beta 3. Thus, these data demonstrate cooperative mechanisms for VPF/VEGF regulation of EC migration involving the alphavbeta3 integrin, the alphavbeta3 ligand OPN, and thrombin cleavage of OPN. These findings also illustrate an operational link between VPF/VEGF induction of EC gene expression and VPF/VEGF enhancement of microvascular permeability, suggesting that these distinct biological activities may act accordingly to stimulate EC migration during angiogenesis.

A delta T-cell receptor deleting element transgenic reporter construct is rearranged in alpha beta but not gamma delta T-cell lineages.

T cells can be divided into two groups on the basis of the expression of either alpha beta or gamma delta T-cell receptors (TCRs). Because the TCR delta chain locus lies within the larger TCR alpha chain locus, control of the utilization of these two receptors is important in T-cell development, specifically for determination of T-cell type: rearrangement of the alpha locus results in deletion of the delta coding segments and commitment to the alpha beta lineage. In the developing thymus, a relative site-specific recombination occurs by which the TCR delta chain gene segments are deleted. This deletion removes all D delta, J delta, and C delta genes and occurs on both alleles. This delta deletional mechanism is evolutionarily conserved between mice and humans. Transgenic mice which contain the human delta deleting elements and as much internal TCR delta chain coding sequence as possible without allowing the formation of a complete delta chain gene were developed. Several transgenic lines showing recombinations between deleting elements within the transgene were developed. These lines demonstrate that utilization of the delta deleting elements occurs in alpha beta T cells of the spleen and thymus. These recombinations are rare in the gamma delta population, indicating that the machinery for utilization of delta deleting elements is functional in alpha beta T cells but absent in gamma delta T cells. Furthermore, a discrete population of early thymocytes containing delta deleting element recombinations but not V alpha-to-J alpha rearrangements has been identified. These data are consistent with a model in which delta deletion contributes to the implementation of a signal by which the TCR alpha chain locus is rearranged and expressed and thus becomes an alpha beta T cell.

Message of nexin 1, a serine protease inhibitor, is accumulated in the follicular papilla during anagen of the hair cycle.

A group of specialized mesenchymal cells located at the root of the mammalian hair follicle, known as the follicular or dermal papillary cells, are involved in regulating the hair cycle, during which keratinocytes of the lower follicle undergo proliferation, degeneration and regrowth. Using the arbitrarily primed-PCR approach, we have identified a 1.3 kb messenger RNA that is present in large quantities in cultured rat follicular papillary cells, but not in skin fibroblasts. This mRNA encodes nexin 1, a potent protease inhibitor that can inactivate several growth-modulating serine proteases including thrombin, urokinase and tissue plasminogen activator. In situ hybridization showed that nexin 1 message is accumulated in the follicular papilla cells of anagen follicles, but is undetectable in keratinocytes or other skin mesenchymal cells. In addition, nexin 1 message level varies widely among several immortalized rat vibrissa papillary cell lines, and these levels correlate well with the reported abilities of these cell lines to support in vivo follicular reconstitution. These results suggest a possible role of nexin 1 in regulating hair follicular growth.

Immortalized rat whisker dermal papilla cells cooperate with mouse immature hair follicle buds to activate type IV procollagenases in collagen matrix coculture: correlation with ability to promote hair follicle development in nude mouse grafts.

An in vivo nude mouse graft model and an in vitro collagen matrix culture system were used to study interactions of immature hair follicle buds from newborn mice with clonally derived AdE1A-12S-immortalized rat whisker dermal papilla cell lines. Of the 19 available dermal papilla cell lines, four consistently supported good hair follicle development and hair growth in grafts. Seven cell lines were clearly negative in this assay, and the remaining eight cell lines yielded poor to moderate hair growth. As a correlate to in vivo extracellular matrix remodeling accompanying hair follicle development, type IV collagenase activity in the medium from cocultures of dermal papilla cells and hair follicle buds was analyzed by gelatin zymography. Hair follicle buds cultured alone secrete primarily the 92-kDa type IV procollagenase. Cocultivation of hair follicle buds with eight of the dermal papilla cell lines resulted in activation of this proenzyme and activation of the 72-kDa and 92-kDa type IV procollagenases produced by the dermal papilla cells. Seven of these eight dermal papilla cell lines support hair growth in the graft system. In the absence of dermal papilla cells, several growth factors induced activation of the 92-kDa procollagenase secreted by hair follicle buds cultured in serum-free medium: epidermal growth factor, transforming growth factor alpha, acidic fibroblast growth factor, and keratinocyte growth factor. The current working hypothesis is that a) hair follicle epithelial cells interact with dermal papilla cells in coculture by mutual induction of growth factors and cytokines that stimulate the release and activation of matrix remodeling proteases; and b) the ability of dermal papilla cells to interact with hair follicle epithelial cells in this way may be crucial for controlled dermal matrix remodelling during HF development.

Perlecan is a component of cartilage matrix and promotes chondrocyte attachment.

Aggrecan, a chondroitin/keratan sulfate-containing proteoglycan, is a major component of cartilaginous tissues. Immunolocalization studies, using antibodies directed to perlecan, a heparan sulfate proteoglycan first detected in basement membranes, and laminin (another major component of basement membranes), indicate that perlecan and laminin are also present in the matrices of hyaline cartilage in the nasal septum, the articular surface of the bone and the growth plate of the developing bone. Consequently, we used antibodies to both aggrecan and perlecan to characterize their synthesis and secretion by primary cultures of chondrocytes derived from the rat chondrosarcoma. Chondrocytes were pulsed for 20 minutes with [35S]methionine and then chased for up to six hours. The radiolabeled perlecan and aggrecan were immunoprecipitated and analyzed by SDS-PAGE. The results show that chondrocytes synthesize precursor proteins to both proteoglycans, but that only the aggrecan precursor protein is secreted as a proteoglycan. Perlecan was also secreted but with less posttranslational modifications than aggrecan. Northern blot analyses of the RNAs from immortalized rat chondrocytes indicated that the major mRNA encoding for perlecan was approximately 13 kb in length, similar in size to that expressed by other cell types, which synthesize 400 kDa core protein perlecan. Analyses of the proteoglycan fractions from the extracts of bovine articular surface indicated that perlecan in this tissue contains both chondroitin and heparan sulfate side-chains. Purified perlecan and laminin were found to promote attachment of immortalized rat chondrocytes in vitro. These studies indicated that perlecan, once thought to be a unique component of the basement membranes, is more widely distributed and is an important component of the cartilage matrix, where it may provide for cell adhesion to the matrix.

Keratinocyte-derived vascular permeability factor (vascular endothelial growth factor) is a potent mitogen for dermal microvascular endothelial cells.

Expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is markedly increased in the epidermis of lesional psoriatic skin and in healing skin wounds. In this study, we characterized the effects of several cytokines and growth factors on the expression and secretion of VPF/VEGF mRNA and protein by cultured human epidermal keratinocytes, as well as the effect of VPF/VEGF on the growth of cultured human dermal microvascular endothelial cells. Transforming growth factor-alpha, epidermal growth factor, and phorbol myristate acetate markedly stimulated VPF/VEGF mRNA expression by cultured keratinocytes; as in psoriatic skin, the three most common VPF/VEGF isoforms (encoding proteins of 121, 165, and 189 amino acids) were upregulated to an equal extent. Transforming growth factor (TGF)-alpha, epidermal growth factor, and phorbol myristate acetate also enhanced the secretion of VPF/VEGF by keratinocytes; in contrast, a number of other cytokines including interleukin (IL)-1, IL-6, IL-8, tumor necrosis factor-alpha, interferon-gamma, and transforming growth factor-beta did not induce VPF/VEGF secretion. The VPF/VEGF secreted by keratinocytes was biologically active in that, like recombinant human VPF/VEGF, it potently stimulated dermal endothelial cell proliferation. Scatchard analysis revealed two high-affinity VPF/VEGF binding sites on dermal endothelial cells with dissociation constants of 51 pM and 2.9 pM. These results suggest that the avascular epidermis has the capacity to regulate dermal angiogenesis and microvascular permeability by a paracrine mechanism involving the secretion of VPF/VEGF. Similar mechanisms may be anticipated in a variety of inflammatory and neoplastic skin diseases characterized by microvascular hyperpermeability, edema, and angiogenesis.

CXC chemokines connective tissue activating peptide-III and neutrophil activating peptide-2 are heparin/heparan sulfate-degrading enzymes.

Heparan sulfate proteoglycans at cell surfaces or in extracellular matrices bind diverse molecules, including growth factors and cytokines, and it is believed that the activities of these molecules may be regulated by the metabolism of heparan sulfate. In this study, purification of a heparan sulfate-degrading enzyme from human platelets led to the discovery that the enzymatic activity residues in at least two members of the platelet basic protein (PBP) family known as connective tissue activating peptide-III (CTAP-III) and neutrophil activating peptide-2. PBP and its N-truncated derivatives, CTAP-III and neutrophil activating peptide-2, are CXC chemokines, a group of molecules involved in inflammation and wound healing. SDS-polyacrylamide gel electrophoresis analysis of the purified heparanase resulted in a single broad band at 8-10 kDa, the known molecular weight of PBP and its truncated derivatives. Gel filtration chromatography of heparanase resulted in peaks of activity corresponding to monomers, dimers, and tetramers; these higher order aggregates are known to form among the chemokines. N-terminal sequence analysis of the same preparation indicated that only PBP and truncated derivatives were present, and commercial CTAP-III from three suppliers had heparanase activity. Antisera produced in animals immunized with a C-terminal synthetic peptide of PBP inhibited heparanase activity by 95%, compared with activity of the purified enzyme in the presence of the preimmune sera. The synthetic peptide also inhibited heparanase by 95% at 250 microM, compared to the 33% inhibition of heparanase activity by two other peptides. The enzyme was determined to be an endoglucosaminidase, and it degraded both heparin and heparan sulfate with optimal activity at pH 5.8. Chromatofocusing of the purified heparanase resulted in two protein peaks: an inactive peak at pI7.3, and an active peak at pI 4.8-5.1. Sequence analysis showed that the two peaks contained identical protein, suggesting that a post-translational modification activates the enzyme.

The tetracycline analogs minocycline and doxycycline inhibit angiogenesis in vitro by a non-metalloproteinase-dependent mechanism.

The tetracycline analogs minocycline and doxycycline are inhibitors of metalloproteinases (MMPs) and have been shown to inhibit angiogenesis in vivo. To further study the mechanism of action of these compounds we tested them in an in vitro model of angiogenesis: aortic sprouting in fibrin gels. Angiogenesis was quantitated in this system by a unique application of planar morphometry. Both compounds were found to potently inhibit angiogenesis in this model. To further characterize the activity of these compounds against MMPs, we determined the IC50S of both compounds against representatives of three classes of metalloproteinases: fibroblast collagenase, stromelysin, and gelatinase A. Doxycycline was found to inhibit collagenase, gelatinase A and stromelysin with IC50S of 452 microM, 56 microM and 32 microM, respectively. Minocycline was found to inhibit only stromelysin in the micromolar range with an IC50 of 290 microM. Since these results suggest that these compounds may not have been inhibiting in vitro angiogenesis by an MMP-dependent mechanism, we decided to test the effects of the potent MMP inhibitor BB-94. This compound failed to inhibit aortic sprouting in fibrin gels, thus strongly suggesting that both doxycycline and minocycline act by an MMP-independent mechanism. These results have implications for the mechanism of action of tetracycline analogs, particularly where they are being considered for the treatment of disorders of extracellular matrix degradation including periodontal disease, arthritis, and tumor angiogenesis.

In vivo regulation of murine hair growth: insights from grafting defined cell populations onto nude mice.

The nude mouse graft model for testing the hair-forming ability of selected cell populations has considerable potential for providing insights into factors that are important for hair follicle development and proper hair formation. We have developed a minimal component system consisting of immature hair follicle buds from newborn pigmented C57BL/6 mice and adenovirus E1A-immortalized rat vibrissa dermal papilla cells. Hair follicle buds contribute to formation of hairless skin when grafted alone or with Swiss 3T3 cells, but produce densely haired skin when grafted with a fresh dermal cell preparation. The fresh dermal cell preparation represents the single cell fraction after hair follicles have been removed from a collagenase digest of newborn mouse dermis. It provides dermal papilla cells, fibroblasts, and possibly other important growth factor-producing cell types. Rat vibrissa dermal papilla cells supported dense hair growth at early passage in culture but progressively lost this potential during repeated passage in culture. Of 19 E1A-immortalized, clonally derived rat vibrissa dermal papilla cell lines, the four most positive clones supported hair growth to the extent of approximately 200 to 300 hairs per 1-2 cm2 graft area. The remaining clones were moderately positive (five clones), weakly positive (three clones), or negative (seven clones). Swiss 3T3 cells prevented contraction of the graft area but did not appear to affect the number of hairs in the graft site produced by dermal papilla cells plus hair follicle buds alone. The relatively low hair density (estimated 1-5% of normal) resulting from grafts of hair follicle buds with the most positive of the immortalized dermal papilla cell clones compared to fresh dermal cells suggests that optimal reconstitution of hair growth requires some function of dermal papilla cells partially lost during the immortalization process and possibly the contribution of other cell types present in the fresh dermal cell preparation, which is not supplied by the Swiss 3T3 cells. The current graft system, comprising hair follicle buds and immortalized dermal papilla cell clones, provides an assay for positive or negative influences on hair growth exerted by added selected cell types, growth factors, or other substances. Characterization of the phenotype of the dermal papilla cell lines, which differ in their ability to support hair growth when grafted with hair follicle buds, may provide insight into specific dermal papilla cell properties important for their function in this system.