Faculty Bibliography

BACKGROUND: The recent discovery of circulating endothelial progenitor cells (EPCs) has altered our understanding of new blood vessel growth such as occurs during collateral formation. Because diabetic complications occur in conditions in which EPC contributions have been demonstrated, EPC dysfunction may be important in their pathophysiology. METHODS AND RESULTS: EPCs were isolated from human type II diabetics (n=20) and age-matched control subjects (n=20). Proliferation of diabetic EPCs relative to control subjects was decreased by 48% (P<0.01) and inversely correlated with patient levels of hemoglobin A1C (P<0.05). Diabetic EPCs had normal adhesion to fibronectin, collagen, and quiescent endothelial cells but a decreased adherence to human umbilical vein endothelial cells activated by tumor necrosis factor-alpha (TNF-alpha) (P<0.05). In a Matrigel assay, diabetic EPCs were 2.5 times less likely to participate in tubule formation compared with controls (P<0.05). CONCLUSIONS: These findings suggest that type II diabetes may alter EPC biology in processes critical for new blood vessel growth and may identify a population at high risk for morbidity and mortality after vascular occlusive events

BACKGROUND: Interaction between hair cells and acellular gels of the mammalian inner ear, the tectorial and otoconial membranes, is crucial for mechanoreception. Recently, otoancorin was suggested to be a mediator of gel attachment to nonsensory cells, but the molecular components of the interface between gels and sensory cells remain to be identified. HYPOTHESIS: We report that the inner ear protein stereocilin is related in sequence to otoancorin and, based on its localisation and predicted GPI-anchoring, may mediate attachment of the tectorial and otoconial membranes to sensory hair bundles. TESTING: It is expected that antibodies directed against stereocilin would specifically label sites of contact between sensory hair cells and tectorial/otoconial membranes of the inner ear. IMPLICATIONS: Our findings support a unified molecular mechanism for mechanotransduction, with stereocilin and otoancorin defining a new protein family responsible for the attachment of acellular gels to both sensory and nonsensory cells of the inner ear.

The wiring of synaptic circuitry during development is remarkably precise, but the molecular interactions that enable such precision remain largely to be defined. Cadherins are cell adhesion molecules hypothesized to play roles in axon growth and synaptic targeting during development. We previously showed that N-cadherin localizes to ventrobasal (VB) thalamocortical synapses in rat somatosensory (barrel) cortex during formation of the whisker-map in layer IV (Huntley and Benson [1999] J. Comp. Neurol. 407:453-471). Such specific association of N-cadherin with one identified afferent pathway raises the prediction that other cadherins are expressed in barrel cortex and that these are, in some combination, also differentially associated with distinct inputs. Here, we first show that N-cadherin and three other classic cadherins (cadherin-6, -8, and -10) are expressed contemporaneously in barrel cortex with relative levels of postnatal expression that are highest during the first 2 weeks, when afferent and intrinsic circuitries are forming and synaptogenesis is maximal. Each displayed distinct, but partly overlapping laminar patterns of expression that changed over time. Cadherin-8 probe hybridization formed a particularly striking pattern of intermittent, columnar patches extending from layer V through layer III, which was first detectable at approximately postnatal day 3. The patches were centered precisely over regions of dysgranular layer IV and, in the whisker barrel field, over barrel septa. This pattern is similar to that formed by the terminal distribution of thalamocortical afferents arising from the posterior nucleus (POm), suggesting cadherin-8 association with the POm thalamocortical synaptic circuit. Consistent with this, cadherin-8 mRNAs were enriched in the POm nucleus, and cadherin-8 immunolabeling in layer IV was enriched in barrel septa and codistributed with labeled POm thalamocortical synaptic-like puncta. The striking molecular parcellation of at least two different cadherins to the two, converging thalamic pathways that terminated in non-overlapping barrel center and septal compartments in layer IV strongly suggested that cadherins provide requisite molecular recognition and targeting that enable precise construction of thalamocortical and other synaptic circuitry.

OBJECTIVE: To study the mechanism of intimal hyperplasia after coronary artery bypass grafting (CABG) and to find an effective way for preventing intimal hyperplasia. METHODS: Twenty-four male New Zealand rabbits were randomly divided into two groups of 12 rabbits: operation group and sham-operation (control) group. The external jugular vein was harvested and anastomosed end-to-side to the ipsilateral carotid artery in operation group or grafted in situ in the control group. Six rabbits in each group were killed and their grafted veins were taken 2 weeks and 4 weeks after operation respectively. The mRNA expressions of transforming growth factor beta (TGF-beta), collagen I, collagen III, and angiotension 1 receptor (AT1R) were measured by RT-PCR and electrophoresis. RESULTS: The intimal hyperplasia was much more remarkable in the operation group than in the control group either 2 weeks or 4 weeks after operation. The mRNA expressions of TGF-beta, AT1R, collagen I, and collagen III were significantly higher in the operation group than in the control group, especially 2 weeks after (P < 0.01). Four weeks after the operation, the expressions of TGF-beta, AT1R, collagen I and collagen III were 4.05 +/- 0.49 vs 2.05 +/- 0.26, 18.23 +/- 1.32 vs 4.61 +/- 0.53, 80 +/- 0.17 vs 0.90 +/- 0.18, and 7.05 +/- 0.68 vs 2.80 +/- 0.17 respectively (all P < 0.05). CONCLUSION: TGF-beta and AT1R may have an important role in the intimal hyperplasia of venous graft in CABG. Continuous arterial pressure may be the main factor of increased expression of TGF-beta and AT1R that cause the enormous synthesis and deposit of collagen

Urothelial plaques consist of four major uroplakins (Ia, Ib, II, and III) that form two-dimensional crystals covering the apical surface of urothelium, and provide unique opportunities for studying membrane protein assembly. Here, we describe a novel 35-kD urothelial plaque-associated glycoprotein that is closely related to uroplakin III: they have a similar overall type 1 transmembrane topology; their amino acid sequences are 34% identical; they share an extracellular juxtamembrane stretch of 19 amino acids; their exit from the ER requires their forming a heterodimer with uroplakin Ib, but not with any other uroplakins; and UPIII-knockout leads to p35 up-regulation, possibly as a compensatory mechanism. Interestingly, p35 contains a stretch of 80 amino acid residues homologous to a hypothetical human DNA mismatch repair enzyme-related protein. Human p35 gene is mapped to chromosome 7q11.23 near the telomeric duplicated region of Williams-Beuren syndrome, a developmental disorder affecting multiple organs including the urinary tract. These results indicate that p35 (uroplakin IIIb) is a urothelial differentiation product structurally and functionally related to uroplakin III, and that p35-UPIb interaction in the ER is an important early step in urothelial plaque assembly

PURPOSE: The majority of the research on the biologic effects of ionizing radiation has focused on the impact of radiation on cells in terms of gene expression, DNA damage, and cytotoxicity. In comparison, little information is available concerning the direct effects of radiation on the extracellular microenvironment, specifically the extracellular matrix and its main component, collagen. We have developed a series of monoclonal antibodies that bind to cryptic epitopes of collagen Type IV that are differentially exposed during matrix remodeling and are key mediators of angiogenesis. We have hypothesized that ionizing radiation might affect the process of angiogenesis through a direct effect on the extracellular matrix and specifically on collagen Type IV. METHODS AND MATERIALS: Angiogenesis was induced in a chick chorioallantoic membrane (CAM) model; 24 h later, a single-dose treatment with ionizing radiation (0.5, 5, and 20 cGy) was administered. Angiogenesis was assessed, and the exposure of two cryptic regulatory epitopes within collagen Type IV (HUI77 and HUIV26) was studied in vitro by solid-phase ELISA and in vivo by immunofluorescence staining. RESULTS: A dose-dependent reduction of angiogenesis with maximum inhibition (85%-90%) occurring at 20 cGy was demonstrated in the CAM model. Exposure of the cryptic HUIV26 site, an angiogenesis control element, was inhibited both in vitro and in vivo by the same radiation dose, whereas little if any change was observed for the HUI77 cryptic epitope. CONCLUSIONS: A dose-dependent alteration of the functional exposure of the HUIV26 cryptic epitope is induced by radiation in vitro and in the CAM model in vivo. This radiation-induced change in protein structure and function may contribute to the inhibitory effects of ionizing radiation on new blood vessel growth and warrants further studies in other models

The Sonic hedgehog (Shh) signaling pathway plays a critical role in normal cerebellar development and has been implicated in medulloblastomas, common malignant childhood tumors of the cerebellum. To test whether Shh mis-expression is sufficient for medulloblastoma formation, we used ultrasound biomicroscopy-guided in utero injection of a Shh-expressing retrovirus into the cerebellum of 13.5-day mouse embryos to show that direct activation of the Shh pathway can lead to tumor formation. Significantly, medulloblastomas were observed in 76% of the mice infected with Shh-expressing retrovirus. Furthermore, contrary to recent suggestions that the Shh transcriptional target Gli1 plays a critical role in Shh-induced tumorigenesis, we found that medulloblastomas form in Gli1 null mutant mice. We have developed an efficient mouse model of medulloblastoma and shown that Gli1 is not required for tumorigenesis when Shh signaling is activated upstream in the pathway

Recent experiments show that hematopoietic progenitor cell populations contain endothelial precursor cells. We have isolated a population of CD34(+) cells that expresses fibroblast growth factor receptor-1 (FGFR-1) and that differentiates into endothelial cells in vitro. We find that 4.5% +/- 2.1% of CD34(+) cells isolated from bone marrow, cord blood, and mobilized peripheral blood express FGFR-1 and that viable CD34(+)FGFR(+) cells are small, with little granularity, and express both primitive hematopoietic and endothelial markers on their surface. The primitive hematopoietic markers AC133, c-kit, and Thy-1 are coexpressed by 75%, 85%, and 64% of CD34(+)FGFR(+) cells, respectively. Most of the CD34(+)FGFR(+) cells also express antigens found on endothelial cells, such as CD31, vascular endothelial growth factor receptor-2, and the endothelial-specific cell surface marker, vascular endothelial cadherin (VE-cadherin), whereas 56% to 60% of the cells express Tie, Tek, and the endothelial-specific marker, P1H12. The CD34(+)FGFR(+) population is enriched in cells expressing endothelial-specific antigens compared with the CD34(+) population. Isolated CD34(+)FGFR(+) cells grow slowly in culture, are stimulated by fibroblast growth factor-2 and vascular endothelial growth factor, and give rise to cells that express von Willebrand factor and VE-cadherin and that incorporate acetylated low-density lipoprotein. These experiments show that FGFR-1 is expressed by a subpopulation of CD34(+) cells that give rise to endothelial cells in vitro, indicating that this population contains endothelial stem/progenitor cells

We have generated an animal model for mitochondrial myopathy by disrupting the gene for mitochondrial transcription factor A (Tfam) in skeletal muscle of the mouse. The knockout animals developed a myopathy with ragged-red muscle fibers, accumulation of abnormally appearing mitochondria, and progressively deteriorating respiratory chain function in skeletal muscle. Enzyme histochemistry, electron micrographs, and citrate synthase activity revealed a substantial increase in mitochondrial mass in skeletal muscle of the myopathy mice. Biochemical assays demonstrated that the increased mitochondrial mass partly compensated for the reduced function of the respiratory chain by maintaining overall ATP production in skeletal muscle. The increased mitochondrial mass thus was induced by the respiratory chain deficiency and may be beneficial by improving the energy homeostasis in the affected tissue. Surprisingly, in vitro experiments to assess muscle function demonstrated that fatigue development did not occur more rapidly in myopathy mice, suggesting that overall ATP production is sufficient. However, there were lower absolute muscle forces in the myopathy mice, especially at low stimulation frequencies. This reduction in muscle force is likely caused by deficient formation of force-generating actin-myosin cross bridges and/or disregulation of Ca(2+) homeostasis. Thus, both biochemical measurements of ATP-production rate and in vitro physiological studies suggest that reduced mitochondrial ATP production might not be as critical for the pathophysiology of mitochondrial myopathy as thought previously