Inter-center variation in death or tracheostomy placement in infants with severe bronchopulmonary dysplasia.

OBJECTIVE: To estimate the presence and sources of inter-center variation (ICV) in the risk of death or tracheostomy placement (D/T) among infants with severe bronchopulmonary dysplasia (sBPD)Study design:We analyzed the Children's Hospitals Neonatal Database between 2010 and 2013 to identify referred infants born <32 weeks' gestation with sBPD. The association between center and the primary outcome of D/T was analyzed by multivariable modeling. Hypothesized diagnoses/practices were included to determine if these explained any observed ICV in D/T. RESULTS: D/T occurred in 280 (20%) of 1383 eligible infants from 21 centers. ICV was significant for D/T (range 2-46% by center, P<0.001) and tracheostomy placement (n=187, range 2-37%, P<0.001), but not death (n=93, range 0-19%, P=0.08). This association persisted in multivariable analysis (adjusted center-specific odds ratios for D/T varied 5.5-fold, P=0.009). CONCLUSIONS: ICV in D/T is apparent among infants with sBPD. These results highlight that the indications for tracheostomy (and subsequent chronic ventilation) remain uncertain.

Predicting death or tracheostomy placement in infants with severe bronchopulmonary dysplasia.

OBJECTIVE: To estimate the risk of death or tracheostomy placement (D/T) in infants with severe bronchopulmonary dysplasia (sBPD) born < 32 weeks' gestation referred to regional neonatal intensive care units. STUDY DESIGN: We conducted a retrospective cohort study in infants born < 32 weeks' gestation with sBPD in 2010-2011, using the Children's Hospital Neonatal Database. sBPD was defined as the need for FiO2 ⩾ 0.3, nasal cannula support >2 l min(-1) or positive pressure at 36 weeks' post menstrual age. The primary outcome was D/T before discharge. Predictors associated with D/T in bivariable analyses (P < 0.2) were used to develop a multivariable logistic regression equation using 80% of the cohort. This equation was validated in the remaining 20% of infants. RESULT: Of 793 eligible patients, the mean gestational age was 26 weeks' and the median age at referral was 6.4 weeks. D/T occurred in 20% of infants. Multivariable analysis showed that later gestational age at birth, later age at referral along with pulmonary management as the primary reason for referral, mechanical ventilation at the time of referral, clinically diagnosed pulmonary hypertension, systemic corticosteroids after referral and occurrence of a bloodstream infection after referral were each associated with D/T. The model performed well with validation (area under curve 0.86, goodness-of-fit χ(2), P = 0.66). CONCLUSION: Seven clinical variables predicted D/T in this large, contemporary cohort with sBPD. These results can be used to inform clinicians who counsel families of affected infants and to assist in the design of future prospective trials.

Hyaluronan serves a novel role in airway mucosal host defense.

Enzymes secreted onto epithelial surfaces play a vital role in innate mucosal defense, but are believed to be steadily removed from the surface by mechanical actions. Thus, the amount and availability of enzymes on the surface are thought to be maintained by secretion. In contrast to this paradigm, we show here that enzymes are retained at the apical surface of the airway epithelium by binding to surface-associated hyaluronan, providing an apical enzyme pool 'ready for use' and protected from ciliary clearance. We have studied lactoperoxidase, which prevents bacterial colonization of the airway, and kallikrein, which mediates allergic bronchoconstriction that limits the inhalation of noxious substances. Binding to hyaluronan inhibits kallikrein, which is needed only in certain situations, whereas lactoperoxidase, useful at all times, does not change its activity. Hyaluronan itself interacts withthe receptor for hyaluronic acid-mediated motility (RHAMM or CD168) that is expressed at the apex of ciliated airway epithelial cells. Functionally, hyaluronan binding to RHAMM stimulates ciliary beating. Thus, hyaluronan plays a previously unrecognized pivotal role in mucosal host defense by stimulating ciliary clearance of foreign material while simultaneously retaining enzymes important for homeostasis at the apical surface so that they cannot be removed by ciliary action.

Respiratory distress after intratracheal bleomycin: selective deficiency of surfactant proteins B and C.

Intratracheal bleomycin in rats is associated with respiratory distress of uncertain etiology. We investigated the expression of surfactant components in this model of lung injury. Maximum respiratory distress, determined by respiratory rate, occurred at 7 days, and surfactant dysfunction was confirmed by increased surface tension of the large-aggregate fraction of bronchoalveolar lavage (BAL). In injured animals, phospholipid content and composition were similar to those of controls, mature surfactant protein (SP) B was decreased 90%, and SP-A and SP-D contents were increased. In lung tissue, SP-B and SP-C mRNAs were decreased by 2 days and maximally at 4--7 days and recovered between 14 and 21 days after injury. Immunostaining of SP-B and proSP-C was decreased in type II epithelial cells but strong in macrophages. By electron microscopy, injured lungs had type II cells lacking lamellar bodies and macrophages with phagocytosed lamellar bodies. Surface activity of BAL phospholipids of injured animals was restored by addition of exogenous SP-B. We conclude that respiratory distress after bleomycin in rats results from surfactant dysfunction in part secondary to selective downregulation of SP-B and SP-C.

Neointimal and tubulointerstitial infiltration by recipient mesenchymal cells in chronic renal-allograft rejection.

BACKGROUND: Tissue remodeling depends on mesenchymal cells (fibroblasts and myofibroblasts) and is a prominent feature of chronic renal-transplant rejection. It is not known whether the mesenchymal cells that participate in remodeling originate locally or from circulating precursor cells. METHODS: We obtained biopsy specimens of renal allografts from six male recipients of an allograft from a female donor, four female recipients of an allograft from a male donor, two male recipients of an allograft from a male donor, and two female recipients of an allograft from a female donor. All the allografts were undergoing chronic rejection. All but two specimens were obtained within six months after transplantation. We used immunohistochemical methods to identify mesenchymal cells with smooth-muscle alpha-actin and in situ hybridization to identify mesenchymal cells with Y-chromosome DNA. RESULTS: No Y-chromosome bodies were identified in the case of the two renal-allograft specimens in which both the donor and the recipient were female. In the case of the two renal-allograft specimens in which both the donor and the recipient were male, approximately 40 percent of mesenchymal cells contained a Y-chromosome body. In the case of the six specimens in which the donor was female and the recipient was male, a mean (+/-SD) of 34+/-16 percent of mesenchymal cells in the neointima, 38+/-12 percent of such cells in the adventitia, and 30+/-7 percent of such cells in the interstitium contained the Y-chromosomal marker, indicating that they originated from the recipient rather than the donor. In the case of the four renal-allograft specimens in which the donor was male and the recipient was female, the respective values were 24+/-15 percent, 33+/-9 percent, and 23+/-8 percent, indicating a persistent population of donor mesenchymal cells. CONCLUSIONS: The presence of mesenchymal cells of host origin in the vascular and interstitial compartments of renal allografts undergoing chronic rejection provides evidence that a circulating mesenchymal precursor cell has the potential to migrate to areas of inflammation.

Differential involvement of the hyaluronan (HA) receptors CD44 and receptor for HA-mediated motility in endothelial cell function and angiogenesis.

Hyaluronan (HA), an important glycosaminoglycan constituent of the extracellular matrix, has been implicated in angiogenesis. It appears to exert its biological effects through binding interactions with at least two cell surface receptors: CD44 and receptor for HA-mediated motility (RHAMM). Recent in vitro studies have suggested potential roles for these two molecules in various aspects of endothelial function. However, the relative contribution of each receptor to endothelial functions critical to angiogenesis and their roles in vivo have not been established. We therefore investigated the endothelial expression of these proteins and determined the effects of antibodies against RHAMM and CD44 on endothelial cell (EC) function and in vivo angiogenesis. Both receptors were detected on vascular endothelium in situ, and on the surface of cultured EC. Further studies with active blocking antibodies revealed that anti-CD44 but not anti-RHAMM antibody inhibited EC adhesion to HA and EC proliferation, whereas anti-RHAMM but not CD44 antibody blocked EC migration through the basement membrane substrate, Matrigel. Although antibodies against both receptor inhibited in vitro endothelial tube formation, only the anti-RHAMM antibody blocked basic fibroblast growth factor-induced neovascularization in mice. These data suggest that RHAMM and CD44, through interactions with their ligands, are both important to processes required for the formation of new blood vessels.

Heterocellular gap junctional communication between alveolar epithelial cells.

We analyzed the pattern of gap junction protein (connexin) expression in vivo by indirect immunofluorescence. In normal rat lung sections, connexin (Cx)32 was expressed by type II cells, whereas Cx43 was more ubiquitously expressed and Cx46 was expressed by occasional alveolar epithelial cells. In response to bleomycin-induced lung injury, Cx46 was upregulated by alveolar epithelial cells, whereas Cx32 and Cx43 expression were largely unchanged. Given that Cx46 may form gap junction channels with either Cx43 or Cx32, we examined the ability of primary alveolar epithelial cells cultured for 6 days, which express Cx43 and Cx46, to form heterocellular gap junctions with cells expressing other connexins. Day 6 alveolar epithelial cells formed functional gap junctions with other day 6 cells or with HeLa cells transfected with Cx43 (HeLa/Cx43), but they did not communicate with HeLa/Cx32 cells. Furthermore, day 6 alveolar epithelial cells formed functional gap junction channels with freshly isolated type II cells. Taken together, these data are consistent with the notion that type I and type II alveolar epithelial cells communicate through gap junctions compatible with Cx43.

A role for hyaluronan in macrophage accumulation and collagen deposition after bleomycin-induced lung injury.

Elevated concentrations of hyaluronan (HA) are associated with the accumulation of macrophages in the lung after injury. We have investigated the role of HA in the inflammatory and fibrotic responses to lung injury using the intratracheal instillation of bleomycin in rats as a model. After bleomycin-induced lung injury, both HA content in bronchoalveolar lavage (BAL) and staining for HA in macrophages accumulating in injured areas of the lung were maximal at 4 d. Increased HA in BAL correlated with increased locomotion of isolated alveolar macrophages. HA-binding peptide was able to specifically block macrophage motility in vitro. Importantly, systemic administration of HA-binding peptide to rats before injury not only decreased alveolar macrophage motility and accumulation in the lung, but also reduced lung collagen alpha (I) messenger RNA and hydroxyproline contents. We propose a model in which HA plays a critical role in the inflammatory response and fibrotic consequences of acute lung injury.

Bleomycin-induced pulmonary injury in mice deficient in SPARC.

SPARC (secreted protein, acidic and rich in cysteine) is a component of the matrix that appears to regulate tissue remodeling. There is evidence that it accumulates in the lung in the setting of pulmonary injury and fibrosis, but direct evidence of its involvement is only now emerging. We therefore investigated the development of pulmonary fibrosis induced by bleomycin administered either intratracheally or intraperitoneally in mice deficient in SPARC. Bleomycin (0.15 U/mouse) given intratracheally induced significantly more pulmonary fibrosis in mice deficient in SPARC compared with that in wild-type control mice, with the mutant mice demonstrating greater neutrophil accumulation in the lung. However, in wild-type and SPARC-deficient mice given intraperitoneal bleomycin (0.8 U/injection x 5 injections over 14 days), the pattern and severity of pulmonary fibrosis, as well as the levels of leukocyte recruitment, were similar in both strains of mice. These findings suggest that the involvement of SPARC in pulmonary injury is likely to be complex, dependent on several factors including the type, duration, and intensity of the insult. Furthermore, increased neutrophil accumulation in the peritoneal cavity was also observed in SPARC-null mice after acute chemical peritonitis. Together, these data suggest a possible role for SPARC in the recruitment of neutrophils to sites of acute inflammation.

Duplication of chromosome region 4q28.3-qter in monozygotic twins with discordant phenotypes.

We describe monozygotic twins with partially discordant phenotypes who were found to have a duplication of chromosome region 4q28.3-qter. The duplicated region of chromosome 4 resulted from an unbalanced segregation of a balanced maternal (4;22)(q28.3;p13) translocation. Duplication of the long arm of chromosome 4 has been described in >60 patients; however, it usually results from the unbalanced segregation of a parental balanced translocation and has an associated monosomy. Twenty cases of dup 4q without an associated monosomy have been reported, and this is the only case of dup 4q28. 3-qter. All cases of dup 4q are reviewed, and phenotypic aspects are analyzed. Issues of monozygotic twinning and other birth defects also are addressed.

Serum hyaluronan and its association with unfavorable histology and aggressiveness of heterotransplanted Wilms' tumor.

BACKGROUND/PURPOSE: The sera and urine of children with Wilms' tumor (WT) often contain increased concentrations of hyaluronan (HA). The authors developed a heterotransplant model to investigate whether serum HA concentrations could predict the histology and progression of WT. METHODS: Random portions of 8 human WT specimens (7 favorable and 1 unfavorable histology findings) were heterotransplanted into the flanks of severe combined immunodeficient (SCID) mice. After 6 to 20 weeks of observation, animals were killed, and serum HA concentrations, tumor histology, and local invasion were determined. RESULTS: Sera of mice supporting tumor growth had a median HA concentration of 9,379 microg/L (range, 459 to 3,206,176 microg/L) compared with a median HA concentration of 416 microg/L (range, 204 to 782 microg/L) in animals not supporting tumor growth. The highest serum HA concentrations were detected in animals harboring unfavorable histology blastemal-predominant tumors, whereas animals supporting favorable histology epithelial- and stromal-predominant tumors had the lowest serum HA concentrations. In association with markedly increased serum HA, undifferentiated blastemal tumors showed significantly greater growth rates than the more differentiated epithelial or stromal tumors. Additionally, serum HA concentrations were greater in mice with invasive as compared with noninvasive tumors for each histological type. Complete resection of established tumors also resulted in the return of serum HA to preheterotransplant concentrations. Identification of tumor progression was further tested in SCID mice receiving subcutaneous flank injections of the human WT cell line, SK-NEP-1. Significantly greater serum HA concentrations again corresponded with more rapid growth rates and invasiveness. CONCLUSIONS: Serum HA concentrations predict the growth, invasion, and unfavorable histology findings of WT in a heterotransplant model. The authors further speculate that HA may foster an environment conducive to WT aggressiveness.

Hyaluronan receptor expression increases in fetal excisional skin wounds and correlates with fibroplasia.

BACKGROUND/PURPOSE: The midgestation fetus heals incisional skin wounds scarlessly, whereas large excisional wounds scar. High concentrations of hyaluronan (HA) are associated with scarless fetal as opposed to scar-forming adult wound repair. Because expression of the HA receptors, CD44 and RHAMM (Receptor for HA-Mediated Motility), has been associated with adult wound fibroplasia, the authors postulated that fetal excisional wounds would show increased expression of CD44 and RHAMM as compared with incisional wounds. METHODS: Two models of fetal wound healing were examined. Fetal skin from human abortuses was heterotransplanted subcutaneously into severe combined immunodeficient (SCID) mice. Fourteen days after grafting, incisional or 2-mm excisional wounds were created (n = 6 per time-point). In addition, incisional and excisional (6 to 10 mm) wounds (n = 5 per time-point) were created on the backs of 70- to 75-day fetal lambs (term, 145 days). Tissue from both models was harvested at sequential time-points after injury. Wounds were studied histologically for fibroplasia and assayed for their HA content. CD44 and RHAMM expression were analyzed by immunohistochemistry and immunoblotting. RESULTS: As expected, in both models, incisional wounds healed scarlessly, whereas excisional wounds showed fibroplasia. Incisional wounds of fetal lambs maintained a significantly higher HA content than excisional wounds 3 days after injury. Between 1 and 7 days in either human or sheep fetal wounds, immunostaining for CD44 and RHAMM markedly increased along the margins of excisional wounds as compared with incisional wounds and unwounded skin. Immunoblot analysis confirmed this increased HA receptor expression in both models. CONCLUSIONS: HA receptor expression increased in both human and sheep fetal excisional wounds and correlated with fibroplasia and a reduced HA content. The authors speculate that strategies to limit the expression or function of HA receptors during postnatal wound repair may modify the development of scar.

Modulation of monocyte-endothelial cell interactions by platelet microparticles.

Platelets, activated by various agonists, produce microparticles (MP) from the plasma membrane, which are released into the extracellular space. Although the mechanism of MP formation has been clarified, their biological importance remains ill defined. We have recently shown that platelet-derived MP influence platelet and endothelial cell function. In this study, we have further examined the mechanism of cellular activation by platelet MP. To address the possibility that they may influence monocyte-endothelial interactions, we used an in vitro assay to examine their effects on the adhesion of monocytes to human umbilical vein endothelial cells (HUVEC). Platelet MP increased the adhesion of monocytes to HUVEC in a time- and dose-dependent manner. Maximal adhesion of monocytes to resting HUVEC was observed after 24 h of stimulation with MP. Similar kinetics were observed with U-937 (human promonocytic leukemia) cells, used as a model for the blood-borne monocyte. Maximal adhesion of resting monocytes to MP-stimulated HUVEC was observed after 5 h of stimulation with MP. The EC50s for MP-induced increases in HUVEC, monocyte, and U-937 cell adhesion is 8.74, 43.41, and 10.83 microg/ml of MP protein, respectively. The induction of monocyte-endothelial adhesion was mimicked by arachidonic acid isolated from MP. The observed increased cellular adhesiveness correlated with MP-induced upregulation of cell adhesion molecules. MP-stimulated HUVEC increased intracellular cell adhesion molecule-1 (ICAM-1) but not vascular cell adhesion molecule-1 (VCAM-1), P-, or E-selectin expression. Monocyte and U-937 lymphocyte function-associated antigen-1 (CD11a/CD18) and macrophage antigen-1 (CD11b/ CD18, alpham/beta2) were both upregulated upon MP stimulation, but an increase in p150,95 (CD11c/CD18), very late antigen-1, or ICAM-1 expression was not observed. The functional importance of these changes was demonstrated with blocking antibodies. MP also induced the chemotaxis of U-937 cells in a dose-dependent manner with an EC50 of 4.40 microg/ml of MP protein. Similarly, arachidonic acid isolated from MP mimicked the chemotactic response. A role for PKC was implicated in both adhesion and chemotaxis. GF 109203X, a specific inhibitor of PKC, significantly reduced monocyte-endothelial adhesion, as well as U-937 chemotaxis. The demonstration that platelet MP may modulate important aspects of endothelial and monocyte function provides a novel mechanism by which platelets may interact with such cells in human atherosclerosis and inflammation.

Overexpression of the hyaluronan receptor RHAMM is transforming and is also required for H-ras transformation.

Overexpression of the RHAMM gene by transfection into fibroblasts is transforming and causes spontaneous metastases in the lung. H-ras-transformed fibrosarcomas transfected with a dominant suppressor mutant of RHAMM exhibit a so-called revertant phenotype and are completely nontumorigenic and nonmetastatic. Conversely, fibroblasts stably expressing low levels of RHAMM as a result of antisense transfection are resistant to ras transformation. Collectively, these results indicate that RHAMM acts downstream of ras. The loss of functional RHAMM ablates signaling within focal adhesions, in particular changes in focal adhesion kinase phosphorylation, and as a result these focal adhesions are unable to turn over in response to hyaluronan. These results provide evidence of the oncogenic potential of a novel extracellular matrix receptor and establish a functional link between transformation by ras and signaling within focal adhesions that are required for transformation by this oncogene.

Migration of bovine aortic smooth muscle cells after wounding injury. The role of hyaluronan and RHAMM.

The migration of smooth muscle cells is a critical event in the pathogenesis of vascular diseases. We have investigated the role of hyaluronan (HA) and the hyaluronan receptor RHAMM in the migration of adult bovine aortic smooth muscle cells (BASMC). Cultured BASMC migrated from the leading edge of a single scratch wound with increased velocity between 1 and 24 h. Polyclonal anti-RHAMM antisera that block HA binding with this receptor abolished smooth muscle cell migration following injury. HA stimulated the random locomotion of BASMC and its association with the cell monolayer increased following wounding injury. Immunoblot analysis of wounded monolayers demonstrated a novel RHAMM protein isoform that appeared within one hour after injury. At the time of increased cell motility after wounding, FACS analysis demonstrated an increase in the membrane localization in approximately 25% of the cell population. Confocal microscopy of injured monolayers confirmed that membrane expression of this receptor was limited to cells at the wound edge. Collectively, these data demonstrate that RHAMM is necessary for the migration of smooth muscle cells and that expression and distribution of this receptor is tightly regulated following wounding of BASMC monolayers.

The role of hyaluronan and its receptors in restenosis after balloon angioplasty: development of a potential therapy.

Atherosclerosis is a progressive condition that is initiated by endothelial injury, promoted by growth factors, and which results in the formation of fibrofatty plaques that narrow the affected blood vessel. Balloon angioplasty is used to dilate these plaques in the coronary circulation so as to prevent occlusion of this critical blood supply. However, 30-50% of balloon dilatations end in restenosis within six months of the procedure. The pathogenesis of both atherosclerosis and restenosis after balloon angioplasty involves the migration of medial smooth-muscle cells across the internal elastic lamina to form a neointima. Proliferation of these cells and their elaboration of an extracellular matrix results in stenosis of the affected area. Investigation of several animal models, as well as of the human condition, indicates the presence of an ongoing inflammatory reaction involving T cells and other leukocytes which probably maintain smooth-muscle cell migration, proliferation and matrix deposition. We have shown that the stenotic response involves the expression of HA (hyaluronan) receptors on both the infiltrating white cells and on smooth-muscle cell populations. Thus, in vitro, the locomotion and chemotaxis of these cells in response to injury is inhibited by reagents that block HA-receptor interactions including HA-binding peptides and high doses of HA. Further, the expression of these HA receptors is up-regulated after balloon-catheter injury of the rat carotid artery, and exposure of injured arteries to high concentrations of HA in vivo results in significant inhibition of neointimal formation. The possible clinical benefits of this response are discussed.