Delayed branching of endothelial capillary-like cords in glycated collagen I is mediated by early induction of PAI-1.

Development of micro- and macrovascular disease in diabetes mellitus (DM) warrants a thorough investigation into the repertoire of endothelial cell (EC) responses to diabetic environmental cues. Using human umbilical vein EC (HUVEC) cultured in three-dimensional (3-D) native collagen I (NC) or glycated collagen I (GC), we observed capillary cord formation that showed a significant reduction in branching when cells were cultured in GC. To gain insight into the molecular determinants of this phenomenon, HUVEC subjected to GC vs. NC were studied using a PCR-selected subtraction approach. Nine different genes were identified as up- or downregulated in response to GC; among those, plasminogen activator inhibitor-1 (PAI-1) mRNA was found to be upregulated by GC. Western blot analysis of HUVEC cultured on GC showed an increase in PAI-1 expression. The addition of a neutralizing anti-PAI-1 antibody to HUVEC cultured in GC restored the branching pattern of formed capillary cords. In contrast, supplementation of culture medium with the constitutively active PAI-1 reproduced defective branching patterns in HUVEC cultured in NC. Ex vivo capillary sprouting in GC was unaffected in PAI-1 knockout mice but was inhibited in wild-type mice. This difference persisted in diabetic mice. In conclusion, the PCR-selected subtraction technique identified PAI-1 as one of the genes characterizing an early response of HUVEC to the diabetic-like interstitial environment modeled by GC and responsible for the defective branching of endothelial cells. We propose that an upregulation of PAI-1 is causatively linked to the defective formation of capillary networks during wound healing and eventual vascular dropout characteristic of diabetic nephropathy.

The Y-box binding protein YB-1 suppresses collagen alpha 1(I) gene transcription via an evolutionarily conserved regulatory element in the proximal promoter.

Appropriate expression of collagen type I, a major component of connective tissue matrices, is dependent on tight transcriptional control and a number of trans-activating and repressing factors have been characterized. Here we identify the Y-box binding protein-1 (YB-1) as a novel repressor of the collagen type alpha1(I) (COL1A1) gene. Collagen type I mRNA and protein levels decreased upon overexpression of YB-1 by transfection in NRK fibroblasts. The human, rat, and mouse COL1A1 promoter -220/+115 contains three putative Y-boxes, one of these sites, designated collagen Y-box element (CYE), includes a Y-box plus an adjacent 3' inverted repeat. DNase-I footprinting and Southwestern blotting with fibroblast nuclear extract demonstrated binding of several nuclear proteins across the CYE, one of which was identified as YB-1. Recombinant YB-1 bound the CYE sequence in gel shift assays with a preference for single-stranded templates. The entire sequence (-88/-48) was required for high affinity binding. Complex formation of endogenous YB-1 with the CYE was established by supershift studies. COL1A1 promoter-reporter constructs were suppressed up to 80% by cotransfection with YB-1 in a variety of cell types. In addition, CYE conferred YB-1 responsiveness on two heterologous promoters further demonstrating the importance of this repressor region. Mung bean nuclease sensitivity analysis suggested that repression is most likely exerted through changes in DNA conformation.

Hypoxia promotes fibrogenesis in human renal fibroblasts.

BACKGROUND: The mechanisms underlying progressive renal fibrosis are unknown, but the common association of fibrosis and microvascular loss suggests that hypoxia per se may be a fibrogenic stimulus. METHODS: To determine whether human renal fibroblasts (HRFs), the primary matrix-producing cells in the tubulointerstitium, possess oxygen-sensitive responses relevant to fibrogenesis, cells were exposed to 1% O2 in vitro. RESULTS: Hypoxia simultaneously stimulated extracellular matrix synthesis and suppressed turnover with increased production of collagen alpha1(I) (Coll-I), decreased expression of collagenase, and increased tissue inhibitor of metalloproteinase (TIMP)-1. These effects are time dependent, require new RNA and protein synthesis, and are specific to hypoxia. The changes in Coll-I and TIMP-1 gene expression involve a heme-protein O2 sensor and protein kinase- and tyrosine kinase-mediated signaling. Although hypoxia induced transforming growth factor-beta1 (TGF-beta1), neutralizing anti-TGF-beta1-antibody did not block hypoxia-induced Coll-I and TIMP-1 mRNA expression. Furthermore, hypoxic-cell conditioned-medium had no effect on the expression of these mRNAs in naive fibroblasts, suggesting direct effects on gene transcription. Transient transfections identified a hypoxia response element (HRE) in the TIMP-1 promoter and demonstrated HIF-1-dependent promoter activation by decreased ambient pO2. CONCLUSIONS: These data suggest that hypoxia co-ordinately up-regulates matrix production and decreases turnover in renal fibroblasts. The results support a role for hypoxia in the pathogenesis of fibrosis and provide evidence for novel, direct hypoxic effects on the expression of genes involved in fibrogenesis.

Is there a common mechanism for the progression of different types of renal diseases other than proteinuria? Towards the unifying theme of chronic hypoxia.

The question of why chronic renal diseases progress is a topic only recently investigated. Putative causes such as proteinuria do not account for all aspects of progressive renal disease. An alternative mechanism, chronic hypoxia, is proposed that might better explain certain elements of progressive renal disease, but elements of the hypothesis remain subject to further study.

Hypoxia-induced changes in extracellular matrix metabolism in renal cells.

The mechanisms underlying the progressive fibrosis that characterises end-stage renal disease in vivo remain to be established but hypoxia, as a result of microvascular injury and loss, has been suggested to play an important role. In support of this hypothesis, in vitro studies show that hypoxia (1% O(2)) induces a fibrogenic phenotype in human renal tubular endothelia, interstitial fibroblasts and microvascular endothelial cells, simultaneously increasing extracellular matrix (ECM) production and decreasing turnover via effectors on matrix-degrading enzymes and their inhibitors. The effects of hypoxia on ECM metabolism are independent of hypoxia-induced growth factors and are mediated by a haem-protein sensor and activation of both protein kinase C- and tyrosine kinase-mediated signal transduction pathways. De novo gene transcription is regulated by both hypoxia-inducible factor-1-dependent and -independent mechanisms. Further understanding of the molecular mechanisms by which decreased oxygen alters expression of genes involved in ECM metabolism in renal cells may provide new insights into the pathogenesis of fibrosis and identify novel avenues for intervention.

Air embolism during tunneled central catheter placement performed without general anesthesia in children: a potentially serious complication.

Central venous catheters have had an increasingly important role in a variety of patient care situations, including long-term antibiotic therapy, chemotherapy, and nutritional support. The recent past has seen a gradual transition from placement of vascular access catheters by surgeons to placement by interventional radiologists. The interventional radiology service places a majority of the vascular access devices at our children's hospital, including peripherally inserted central catheters, tunneled central venous catheters, temporary and permanent hemodialysis catheters, and subcutaneous ports. Most procedures performed by our interventional radiology service in children can be successfully completed with use of intravenous (i.v.) sedation, and a few require general anesthesia (GA). Key advantages of GA over i.v. sedation include the ability to have positive pressure ventilation (PPV) or controlled apnea during the procedure. We report our experience of venous air embolism in three small children during placement of tunneled central venous catheters when GA was not used.

Progressive renal disease: fibroblasts, extracellular matrix, and integrins.

Progressive renal disease is characterized by expansion of the tubulo-interstitium and accumulation of extracellular matrix within this tissue compartment. Interstitial fibroblasts are the primary producers of the interstitial matrix, and in the evolution of tubulo-interstitial fibrosis these cells undergo changes, namely increased proliferation, differentiation to myofibroblasts, and altered extracellular matrix metabolism, all of which, in other cell types, have been shown to be regulated by the major family of extracellular matrix receptors, the integrins. In the normal kidney, interstitial fibroblasts express alpha1, alpha4, alpha5, and beta1 integrins, and fibrosis is associated with increased expression of alpha1, alpha2, alpha5, alphav, and beta1 integrins. In particular, alpha5, beta1, and alphav are suggested to be linked with the fibrotic process. In vitro, renal fibroblasts express a similar range of integrins, and ligation of selected receptors is associated with specific functions. Ligation of alpha6 stimulates proliferation, while alpha5 promotes expression of myofibroblastic phenotype, and beta1 integrin has been implicated in cell contraction. Recent studies suggest that renal fibroblasts also express the non-integrin matrix receptors, discoidin domain receptors, and that changes in activation of these receptors may be associated with fibrogenic events. Thus the current, albeit limited, data suggest an important role for receptors for extracellular matrix molecules in the pathogenesis of progressive renal fibrosis.

Differential response of activated versus non-activated renal fibroblasts to tubular epithelial cells: a model of initiation and progression of fibrosis?

BACKGROUND/AIMS: The mechanisms of initiation and maintenance of renal fibrosis remain obscure, but one hypothesis highlights the importance of tubular epithelial cell-interstitial fibroblast interactions and suggests that tubular injury may be a precipitating factor. The aims of the study were to examine the effects of factors of proximal tubular origin on renal fibroblasts expressing different levels of smooth muscle actin (SMA; a putative marker of fibroblast activation) and to examine the modulation of SMA by extracellular matrix (ECM) proteins and transforming growth factor beta 1 (TGF-beta 1), a major profibrotic cytokine. METHODS: Primary cultures of rat cortical fibroblasts (CF) and the rat kidney fibroblast cell line NRK-49F were (1) cultured on different ECM proteins; (2) treated with medium conditioned by rat proximal tubular epithelial cells (PTE), and (3) treated with TGF-beta 1. SMA protein expression was examined by immunocytochemistry, while expression of MMP-2, MMP-3, TIMP-1, and collagen I mRNA was determined by Northern blot analysis or reverse-transcriptase polymerase chain reaction. RESULTS: In cells with low basal levels of SMA (CF), serum was the most potent inducer of increased SMA expression, although ECM proteins also modulated expression. With high basal levels of SMA expression (NRK), ECM proteins alone had little or no effect, but acted synergistically with serum to stimulate expression. In CF, PTE-conditioned medium (CM) had no effect on SMA and TIMP mRNA levels, but suppressed expression of MMP mRNAs. In NRK-49F, PTE-CM stimulated SMA and TIMP-1 mRNA levels, but had no effect on MMP mRNA levels. Although TGF-beta 1 modulated some cellular responses in NRK-49F, neutralizing antibody studies showed it was not the main mediator of the PTE-CM-induced effects. CONCLUSIONS: These data suggest (1) that in renal fibroblasts SMA expression can be modulated by both serum and ECM proteins and (2) that PTE induce a fibrogenic phenotype in both non-activated (low SMA) and activated (high SMA) fibroblasts via different mechanisms.

Umbilical metastasis (Sister Mary Joseph's nodule) in a child.

Metastatic malignancy to the umbilicus, also known as Sister Mary Joseph's nodule, is a rare condition, which to our knowledge has not been reported in a child. We present a case of an umbilical metastasis from carcinoma of the colon in a boy who was also receiving chemotherapy for mediastinal lymphoblastic lymphoma.

Hypoxia stimulates proximal tubular cell matrix production via a TGF-beta1-independent mechanism.

Tubulointerstitial fibrosis is characterized by tubular basement membrane thickening and accumulation of interstitial extracellular matrix (ECM). Since chronic low-grade hypoxia has been implicated in the pathogenesis of fibrosis and proximal tubular epithelial cells (PTE) are sensitive to oxygen deprivation, we hypothesized that hypoxia may stimulate ECM accumulation. In human PTE, hypoxia (1% O2, 24 hr) increased total collagen production (15%), decreased MMP-2 activity (55% +/- 13%; control = 100%) and increased tissue inhibitor of metalloproteinase-1 (TIMP-1) protein. Collagen IV mRNA levels decreased while collagen I mRNA increased, suggesting induction of interstitial collagen. Hypoxia-induced changes persisted on re-oxygenation with increased expression of TIMP mRNAs. A potential mediator for these effects is transforming growth factor-beta1 (TGF-beta1), a major pro-fibrogenic factor produced by PTE. Although hypoxia stimulated TGF-beta production (2- to 3-fold), neutralizing anti-TGF-beta1 antibody did not abolish the hypoxia-induced changes in gelatinase activity, TIMP-1, collagen IV or collagen I mRNA expression, implying that TGF-beta1 is not the mediator. Furthermore, exogenous TGF-beta1 (0 to 10 ng/ml) did not mimic hypoxia, as it stimulated MMP-2 activity and increased the expression of collagen IV, collagen I and TIMP-1 mRNA. The data suggest that hypoxia may be an important pro-fibrogenic stimulus independent of TGF-beta1.

Acidic FGF regulation of hyperproliferation of fibroblasts in human autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cystic tubule enlargement and expansion of the interstitium associated with fibrosis. Our previous studies have analyzed the increased proliferation of cystic epithelial cells and this study examines the basis of increased proliferation of interstitial fibroblasts associated with ADPKD disease progression. ADPKD fibroblasts show phenotypic alterations in vitro, have acquired the capacity to grow in soft agar, and show an increased mitogenic response to a variety of growth factors particularly acidic FGF (aFGF). ELISA, Western immunoblot analysis, and immunocytochemistry showed increased aFGF content in ADPKD tissues and fibroblasts in culture, and aFGF was secreted into the extracellular matrix and conditioned medium, respectively. No alterations in aFGF receptor number were found, but Scatchard analysis of 125I-aFGF binding suggested an increased affinity of binding to the low affinity receptor, and covalent cross-linking analysis suggested the presence of novel putative receptors (120 kDa) in ADPKD fibroblasts. Signaling abnormalities were found, since aFGF incubation resulted in the tyrosine phosphorylation of additional substrates, more rapidly and for a more sustained duration in ADPKD fibroblasts than in normal fibroblasts. These findings suggest an important role for acidic FGF in the hyperproliferation of interstitial fibroblasts associated with disease progression in human ADPKD.

Strength of fixation of anterior vertebral body screws.

STUDY DESIGN: The technique of hole preparation and the placement or orientation of anterior transvertebral screws in relation to the endplates were the subject of two experiments. OBJECTIVES: Experiment 1--to determine if anterior transvertebral body screws have higher pull-out strengths after unicortical predrilling compared with a bicortical predrilling; Experiment 2--to determine the effect of the screw's orientation in the vertebral body on its resistance to loosening. SUMMARY AND BACKGROUND DATA: There are a variety of surgical techniques for using anterior transvertebral screws in the vertebral body without any clear guidelines as to which techniques optimize the strength of fixation. METHODS: In experiment 1, 31 cadaveric vertebral bodies were tested for pull-out strength of transvertebral body screws placed after unicortical predrilling compared with bicortical predrilling. In experiment 2, 48 cadaveric vertebral bodies were tested with transvertebral screws inserted using four different screw orientations. RESULTS: There is no statistically significant difference in pull-out strength of anterior transvertebral body screws inserted after unicortical compared with bicortical predrilling (Experiment 1). The resistance to loosening is greatest when transvertebral screws are oriented in the "superior oblique" position (Experiment 2). CONCLUSIONS: Experiment 1--preparation of the far cortex by predrilling is not necessary. Experiment 2--transvertebral screws should be obliquely oriented so that the forces applied to the screws are resisted by both of the endplates.

Pexicrine effects of basement membrane components on paracrine signaling by renal tubular cells.

Paracrine interactions between tubular epithelium and interstitial cells have been assumed to be mediated largely by soluble cytokines. While the role of extracellular matrix (ECM) and matrix metalloproteinases (MMPs) in modifying cell function is widely appreciated, the role of the renal tubular basement membrane in modulation of tubulointerstitial function has not been studied. To establish whether those components of the ECM which support tubular epithelial cells also influence cell function (that is, a pexicrine effect), we studied their effects on paracrine signaling between epithelium and fibroblasts. Primary cultures of rat renal proximal tubular epithelial cells (PTE) were cultured on laminin (LN), collagen types-IV and -I (COL-IV, COL-I) and fibronectin (FN). PTE attained confluence more rapidly when grown on LN = COL-IV > COL-I = FN = plastic. On all substrates PTE produced the MMPS, gelatinase-A and -B and collagenase with an apparent increase in gelatinase-A and -B production when cultured on LN. MMPs were found to be secreted both apically and basally with basal secretion predominating, except on LN where secretion was primarily from the apical surface. Cultures of rat renal cortical interstitial fibroblasts were established and characterized. Cortical fibroblasts (CF) were found to secrete gelatinase-A and collagenase. Conditioned medium (CM) from PTE cultured on COL-IV stimulated proliferation of CF but proliferation was unaltered by CM from PTE grown on other substrates. By contrast, co-culture of PTE on LN with CF suppressed collagenase and gelatinase activity in both cell types, indicating a bi-directional, paracrine modulation of MMP production. Thus in the tubulointerstitium, the BM components LN and COL-IV not only fulfill a structural role but act as signaling molecules with differential effects which modify the function of the tubular epithelium and its paracrine interaction with adjacent fibroblasts. The initiation of interstitial fibrosis induced by injury to the tubular basement membrane may reside in the perturbation of this interaction.

A rabbit model for nonunion of lumbar intertransverse process spine arthrodesis.

STUDY DESIGN: A rabbit model for lumbar intertransverse process spine arthrodesis was utilized, with blind evaluation of the control and experimental animals. OBJECTIVES: To establish an animal model for motion-induced nonunion of lumbar intertransverse process spine arthrodesis and to determine if there was an early time period during which excessive motion at the arthrodesis site was deleterious to fusion. SUMMARY OF BACKGROUND DATA: Most previously reported animal models for nonunion of lumbar spine arthrodesis do not use intertransverse process fusion and do not allow variable control of the nonunion-inducing factor. METHODS: Forty-two adult New Zealand white rabbits underwent posterolateral intertransverse process spine arthrodesis with autogenous iliac crest graft. Rabbits in the experimental group (n = 23) underwent a lifting protocol to produce motion at the arthrodesis site; those in the control group (n = 12) remained undisturbed in their cages. Arthrodeses were assessed radiographically and manually to determine if fusion had occurred. RESULTS: Lifted rabbits exhibited a 13% fusion rate compared to a 50% fusion rate in controls. Rabbits lifted during only the first 2 postoperative weeks exhibited a fusion rate that was significantly less (P = 0.03) than that of controls. CONCLUSIONS: A small animal model for motion-induced nonunion of lumbar intertransverse process spine arthrodesis has been established. With respect to excessive motion as a promoter of nonunion in spine arthrodeses, the early postoperative time period appears critical.

The effect of different surgical releases on thoracic spinal motion. A cadaveric study.

STUDY DESIGN: Two separate experiments (A and B), each involving six human cadaveric torsos with intact rib cages and sternums, were done to determine the effect of two different sequences of surgical releases (at T8-T9) on thoracic spinal motion. OBJECTIVES: Experiment A was designed to test the effects of three releases in sequence from anterior to posterior, analogous to a two-stage operative treatment with anterior and posterior releases. Experiment B, which involved three releases, was designed to determine 1) if unilateral posterior total facetectomy alone allowed a significant increase in motion and 2) if rib head resection without discectomy allowed a significant increase in motion. SUMMARY OF BACKGROUND DATA: In the surgical treatment of thoracic spinal deformity, surgical release is often done to impart additional flexibility to the spine. Available releases include discectomy, rib head resection, and facetectomy. There is little work to date on the relative importance of the disc, rib head, and facet joint in the stability of the thoracic spine. METHODS: In experiment A and experiment B, the cadaveric torsos were mounted on a custom-made loading frame. Mechanical testing (using weights, pulleys, and digital goniometers) was done after each surgical release to measure the extent of angular rotation in the coronal plane (right lateral bending and left lateral bending) and in the sagittal plane (flexion and extension). RESULTS: The combination of rib head resection and radical discectomy provided the greatest increase in thoracic spinal motion. Standard discectomy alone did not allow a significant increase in motion. Rib head resection without discectomy did not allow a significant increase in motion. Unilateral posterior total facet excision did not allow a significant increase in motion. CONCLUSIONS: These experiments indicate that the combination of rib head resection and radical discectomy may be the optimal thoracic spinal release.