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.

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.

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.

Vascular permeability factor (vascular endothelial growth factor) in guinea pig and human tumor and inflammatory effusions.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor, is a dimeric M(r) 34,000-42,000 glycoprotein that possesses potent vascular permeability-enhancing and endothelial cell-specific mitogenic activities. It is synthesized by many rodent and human tumor cells and also by some normal cells. Recently we developed a sensitive and specific time-resolved immunofluorometric assay for quantifying VPF in biological fluids. We here report findings with this assay in guinea pigs and patients with both malignant and nonmalignant effusions. Line 1 and line 10 tumor cells were injected into the peritoneal cavities of syngeneic strain 2 guinea pigs, and ascitic fluid, plasma, and urine were collected at various intervals. Within 2 to 4 days, we observed a time-dependent, parallel increase in VPF, ascitic fluid volume, and tumor cell numbers in animals bearing either tumor line; in contrast, VPF was not detected in plasma or urine, even in animals with extensive tumor burdens. However, low levels of VPF were detected in the inflammatory ascites induced by i.p. oil injection. In human studies, high levels of VPF (> 10 pM) were measured in 21 of 32 effusions with cytology-documented malignant cells and in only seven of 35 effusions without cytological evidence of malignancy. Thus, VPF levels in human effusions provided a diagnostic test for malignancy with a sensitivity of 66% and a specificity of 80% (perhaps as high as 97% in that six of the seven cytology-negative patients with VPF levels > 10 pM had cancer as determined by other criteria). As in the animal tumor models, VPF was not detected in serum or urine obtained from patients with or without malignant ascites. Many nonmalignant effusions contained measurable VPF but, on average, in significantly smaller amounts than were found in malignant effusions. VPF levels in such fluids correlated strongly (p = 0.59, P < 0.001) with monocyte and macrophage content. Taken together, these data relate ascitic fluid accumulation to VPF concentration in a well-defined animal tumor system and demonstrate, for the first time, the presence of VPF in human malignant effusions. It is likely that VPF expression by tumor and mononuclear cells contributes to the plasma exudation and fluid accumulation associated with malignant and certain inflammatory effusions. The VPF assay may prove useful for cancer diagnosis as a supplement to cytology, especially in tumors that grow in the pleural lining but not as a suspension in the effusions that they induce.

Localization of myeloperoxidase to the long arm of human chromosome 17: relationship to the 15; 17 translocation of acute promyelocytic leukemia.

Myeloperoxidase (MPO) is an enzyme whose synthesis is restricted to the promyelocytic stage of myeloid differentiation. We have recently described the cloning and sequencing of a cDNA for MPO. Using a regional mapping panel of somatic cell hybrids containing various deleted or translocated segments of chromosome 17, we have assigned MPO to a region between 17q21 and 17q23. In situ hybridization refined this localization in that grains on chromosome 17 were significantly clustered at bands q22-23 and no hybridization was detected at q21. In light of this chromosome assignment, the relationship of MPO to the 17q translocation breakpoint characteristic of acute promyelocytic leukemia (APL) was considered. Because this breakpoint has been variously assigned to different bands on 17q from 17q11.2 to 17q22, the cytogenetic and molecular distance between this breakpoint and MPO cannot be accurately determined. MPO and other probes mapped to this region of 17 will be important in searching for altered Southern blot patterns after conventional or pulsed-field gel analysis of DNA from APL patients.

Altered steady-state mRNA levels of basement membrane proteins in diabetic mouse kidneys and thromboxane synthase inhibition.

We examined steady-state levels of mRNA encoding type IV collagen, B1 chain of laminin, and the basement membrane heparan sulfate proteoglycan in the kidney cortex of a mouse model (KKAy) of non-insulin-dependent diabetes. mRNAs encoding laminin B1 and the proteoglycan were unchanged in kidneys taken from diabetic mice with demonstrable basement membrane thickening. mRNA levels for type IV collagen, in contrast, were significantly elevated (2-fold) in diabetic mice concurrent with but not preceding morphologically thickened basement membranes. There was a negative correlation between a ratio of proteoglycan/type IV collagen and levels of albuminuria in the diabetic mice. No correlation was noted with laminin. We also examined the effects of inhibiting the synthesis of thromboxane, a potent vasoconstrictor, on the steady-state levels of type IV collagen in the diabetic mice. Inhibition of thromboxane stopped the progression of albuminuria and prevented an increase in type IV collagen mRNA levels. We conclude that basement membrane thickening in diabetes, a hallmark of diabetic nephropathy, is partly a consequence of an unbalanced increase in the production of type IV collagen. The relative decrease in proteoglycan production may contribute to chronic albuminuria.

Response of diabetic basement membrane--producing cells to glucose and insulin.

The effect of insulin and glucose on the synthesis of basement membrane components was studied in organ cultures of a basement membrane-producing tumor grown in diabetic and normal mice. Tumor tissue grown in diabetic mice produced more protein and basement membrane-specific proteoglycan in response to insulin than tissue grown in normal mice. Addition of high levels of glucose to the culture medium did not alter insulin-stimulated protein synthesis by diabetic or normal tissue but dampened insulin-stimulated production of proteoglycan. These data suggest that basement membrane-producing cells in diabetic hosts may be hypersensitive to insulin and that stimulation of protein production by insulin may play some role in the in situ hypertrophy of basement membranes.

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.

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.

Regulation of hair follicle development: an in vitro model for hair follicle invasion of dermis and associated connective tissue remodeling.

During embryonic development presumptive hair follicle cells of epithelial and mesenchymal origin are determined in defined body locations. This is followed by rapid proliferation of epithelial cells and associated penetration into the dermis in response to as yet undetermined signals. A collagen matrix culture system, which maintains the three-dimensional relationships of hair follicle cells to each other, was developed to study the regulation of the enlargement of immature hair follicles and the accompanying remodeling of the dermis. In studies with a heterogeneous dermis-derived preparation of murine hair follicles, ranging in size from the earliest down-growing budding cell mass to hair-forming follicles, we had previously shown that cell proliferation was stimulated by cholera toxin and epidermal growth factor, but only the epidermal growth factor-stimulated proliferation was accompanied by digestion of the collagen matrix due to release of collagenolytic enzymes. Further studies revealed that transforming growth factor-alpha also stimulated hair follicle cell proliferation and collagenase release. However, although transforming growth factor-beta inhibited the transforming growth factor-alpha-stimulated proliferation, it enhanced the release and activation of collagenases and other gelatin-degrading enzymes detectable by gelatin zymography. Stimulation of collagenolytic activity depended on the three-dimensional hair follicle structure and did not occur in monolayer cultures of hair follicle cells. Comparison of hair follicle buds with more developed dermis-derived hair follicles, plated at the same cell density (based on DNA content), suggested that a greater fraction of cells in the bud-stage follicle responded to the growth factors by release of collagenases. Possibly only the cells in the advancing portion of growing hair follicles that are closest to the dermal papilla cell cluster produce the collagenases in response to growth factors. To examine the participation of dermal papilla cells in collagenase release and activation, several immortalized rat whisker dermal papilla cell lines were co-cultured with mouse hair follicle buds. Co-culture resulted in a marked enlargement of follicles as well as activation of the 92-kDa type IV collagenase, produced by hair follicle buds, that correlated with ability of the dermal papilla cells to stimulate hair formation in grafts of hair follicle buds on nude mice. Dermal papilla cells cultured alone produced the 72-kDa type IV collagenase, which was also activated during co-culture with hair follicle buds. Thus, two activities, both relevant for hair follicle development, namely, cell proliferation and release and activation of collagenases, have been stimulated in immature hair follicle buds by either growth-factor supplementation or interaction with dermal papilla cells.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

Reconstitution of hair follicle development in vivo: determination of follicle formation, hair growth, and hair quality by dermal cells.

Combinations of cultured and uncultured epidermal and dermal cell preparations from newborn and perinatal mice were grafted onto the backs of athymic nude mouse hosts to elucidate the cellular requirements for skin appendage formation. All epidermal populations studied, including a total epidermal keratinocyte preparation from trypsin-split skin, developing hair follicle buds isolated from epidermis, and preformed hair follicles isolated from dermis, make haired skin when grafted with fresh dermal cells. Only pre-formed hair follicles produce haired skin on grafts without an additional dermal component. Hair follicle buds grafted alone or with cultured dermal cells will reconstitute skin but without appendage formation. Thus, cells or factors present in fresh, but not cultured, dermal cells are essential for supporting hair growth from budding follicles, whereas more developed (pre-formed) follicles appear to contain all the necessary components for hair formation. Dissociation of isolated hair follicles by trypsin/ethylenediaminetetraacetic acid prior to grafting is permissive for hair growth, suggesting that follicle cells can be re-induced or reassociate in vivo. Dermal papilla cells, microdissected from rat vibrissal follicles and cultured for up to 14 passages, stimulate hair growth from follicle buds and influence the quality of hair growth from pre-formed hair follicles. Thus, dermal papilla cells maintain inductive capacity in culture and contribute to the reconstituted skin. This reconstitution model should be useful for identifying cell populations within the hair follicle compartment necessary for hair growth and for examining the effects of specific gene products on hair follicle growth and development in vivo.

Secondary aqueous humor stimulates the proliferation of cultured bovine corneal endothelial cells.

Secondary aqueous humor (2 degrees AH) is known to contain elevated levels of serum macromolecules and has been shown to stimulate the proliferation of lens epithelial cells both in vivo and in vitro as well as corneal endothelial cells in vitro. The purpose of this study was to characterize the response of bovine corneal endothelial cells to 2 degrees AH from rabbits and to compare the effect when the cells were grown on plastic dishes covered with an extracellular matrix or on plastic alone. The addition of varying amounts of 2 degrees AH protein (0.1 to 10 mg/ml) to bovine corneal endothelial cells (cultured in MEM plus 1% serum) resulted in a dose dependent proliferative response as measured by the incorporation of 3H-thymidine into DNA. Except for a 2-hr lag phase, the proliferative response increased with increasing time of exposure (6-18 hrs) of the cells to 2 degrees AH containing a constant amount (2.0 mg/ml) of protein. The generation time and final density of the cells, but not the plating efficiency, was significantly greater when the cells were grown in the presence of 2 degrees AH protein on an extracellular matrix rather than plastic alone. Selective adsorption of prostaglandins and aromatic compounds from 2 degrees AH reduced its ability to produce a proliferative response to control levels. These results indicate that 2 degrees AH can alter or regulate events in the cell cycle of corneal endothelial cells. The responsible factor(s) could be involved in control of cellular regeneration in the eye following injury.

New somatic cell hybrids for physical mapping in distal Xq and the fragile X region.

Somatic cell hybrids were constructed from 3 patients carrying X chromosome abnormalities with breakpoints in distal Xq: 1) 94-3, from a patient with 46,XX,t(X;15)(q25 or q26;q25), 2) 8121-A1, from a patient with 46,X,del(X)(q26), and 3) 2384-A2, from a patient with 46,X,del(X)(q27). The breakpoint of patient 94 is proximal to HPRT in q26, a significant distance from the fragile X locus. The breakpoint of patient 8121 is distal to F9, but proximal to DXS98, and is thus proximal to the fragile site region. The breakpoint of 2384 is distal to DXS98 but proximal to DXS52, placing it within the region of the fragile site. Use of these physical mapping reference points will aid in the rapid localization of new DNA markers to distal Xq and the fragile X region.

Anatomic, functional, and pathologic changes from internal ureteral stent placement.

The anatomic, hydrodynamic, functional, and pathologic changes associated with unilateral internal ureteral stenting were evaluated in 20 female canines. Selective glomerular filtration rates (GFR) were measured with technetium 99m diethylenetriamine pentaacetic acid (DTPA) renal scans (N = 14) prior to and several weeks after unilateral internal stent placement. Cystometry and cystography were done at weekly intervals to determine if reflux occurred and to measure the intravesical pressure to produce this reflux (N = 16). Ureteral lumenal capacities of mid 6-cm ureteral segments of stented and unstented ureters were compared. The mid-ureteral lumenal volumes were three times greater in the stented ureters (p < 0.002). There were no significant differences in the selective GFR before and after stenting. Low-pressure vesicoureteral reflux occurred at a mean intravesical pressure of 13.7 cm of water and was present in 84.6 percent (11/13) of the canines whose stents did not migrate or obstruct from encrustation. There were no significant alterations in serum chemistries or blood counts. Fluoroscopic imaging also showed ineffective ureteral peristalsis. This study confirms that internal ureteral stents cause vesicoureteral reflux and significant lumenal dilation without altering renal function.

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.

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.