Project 1 Update

The 2015 progress report highlighted the effects of heparin in preventing hyperglycemic dividing smooth muscle cells from initiating intracellular synthesis of hyaluronan with resultant autophagy and extrusion of a monocyte-adhesive extracellular matrix after completing division. The stated goal for this past year was to attempt to identify the heparin receptor and to explore the mechanism involved in preventing the intracellular hyaluronan response. A biotinylated heparin was used to capture any binding proteins present on the surface of G0/G1 growth arrested cells. After solubilizing the plasma membrane, streptavidin beads were used to isolate the biotinylated heparin, which bound only 2 proteins identified as calreticulin and prelamin A/C isoform X1.

We also explored glucose uptake in the hyperglycemic dividing cells alone or in the presence of heparin. Within 1 hour after progressing from G0/G1, glucose uptake was blocked by the heparin treatment in contrast to the hyperglycemic cells in the absence of heparin. By 6 hours UDP-glucose in the untreated hyperglycemic cells was greatly increased. In contrast, heparin treated hyperglycemic cells maintained a low level similar to the cells dividing in normal glucose levels. This provides evidence that heparin interaction with the receptor initiates signaling pathways that block glucose uptake, most likely until cell division is completed. Our published studies show that after completing division the hyperglycemic cells treated with heparin synthesize a much larger monocyte-adhesive extracellular hyaluronan matrix than do the cells in hyperglycemic medium alone, which indicates that glucose uptake must be restored after division.

Last year’s progress report also proposed that monocytes/macrophages that are exposed to hyperglycemia will express an M1 pro-inflammatory phenotype and that the presence of heparin will prevent this and promote an M2 phenotype. Our new experiments with human U937 monocytes and monocyte/macrophages provide strong evidence that this is indeed true. Hyperglycemic U937 monocytes treated with PMA to promote a macrophage phenotype expressed cell surface CD80, an M1 marker. When heparin was present, the M1 marker was not expressed while the cell surface M2 marker CD163 was expressed. Murine bone marrow monocytes/macrophages showed similar results; hyperglycemia induced gene expression of the M1 protein iNOS, while heparin prevented its expression and induced gene expression of the M2 protein arginase. This supports our observations that the presence of macrophages in heparin treated diabetic rats appear able to remove the glomerular hyaluronan matrix produced by the mesangial cells without promoting a pro-inflammatory response.

Amina Abbadi, the graduate student in our lab, successfully defended her thesis in September and is now a PhD postdoc working on this project. Her thesis established an air-lift transwell model for airway epithelial cells cultured on a basement membrane to form a pseudo-stratified airway epithelium and showed that it synthesizes a heavy chain 3-hyaluronan (HC3-HA) raft on its ciliated surface. She also showed that BAL from normal mice also contained this modified HC3-HA matrix (J. Biol. Chem. manuscript in minor revision). This HC3-HA matrix was first identified as urinary trypsin inhibitor (UTI) on renal endothelial cells, and is likely to be on vascular endothelial cells as well. Amina will test this with our HUVEC model on the transwell basement membrane model this coming year. Her airlift airway epithelium model is now being used in co-cultures with airway smooth muscle cells to determine mechanisms for cross-talk in several asthma projects in Serpil Erzurum’s NHLBI PPG and TPPG programs indicative of the synergistic interactions of the PEG projects and the importance of Amina’s continued work in the PEG.

I presented invited talks on our work at a European Connective Tissue Research meeting in Cologne, Germany May 1-3, and at the AMSB meeting in Cleveland October 13-15 where I received the Senior Research Award (for my research not my age). Amina Abaddi was invited to give a short talk at the ASBMB meeting, April 26-30 in San Diego.

One objective for the coming year is to use the biotinylated heparin model to determine the proteins bound during the time frame (0-3 hours) for its internalization and transport to the ER, Golgi, nucleus. This will likely identify other cytosolic proteins that participate during heparin’s induction of signaling pathways through its receptor that block the activation of hyaluronan synthase in intracellular membrane compartments and that re-program the cells to synthesize the excessive extracellular hyaluronan matrix after completing division.

Another objective for the coming year is to carefully dissect the time course (0-24 hours) from G0/G1 through cell division for glucose transport and to measure the changes in UDP-glucose, and in the precursors for hyaluronan synthesis, UDP-GlcUA and UDP-GlcNAc, in hyperglycemic medium without or with heparin. We expect that UDP-GlcNAc in hyperglycemic cells will increase following the observed large increase of UDP-glucose at 6 hours and activate the synthesis of hyaluronan in intracellular compartments, which begins at or shortly after entering S phase (~8-10 hours), and that heparin will prevent this.

1) Lauer ME, Aytekin M, Comhair SA, Loftis J, Tian L, Farver CF, Hascall VC, Dweik RA. Modification of hyaluronan by heavy chains of inter-alpha-inhibitor in idiopathic pulmonary arterial hypertension. J Biol Chem. 289:6791-8, 2014. PMCID: PMC3945340.
2) Ghatak S, Misra S, Norris RA, Rodriquez RM, Hoffman S, Levine RA, Hascall VC, Markwald RR. Periostin induces intracellular cross talk between kinases and hyaluronan in atrioventricular valvulogenesis. J Biol Chem. 289:8545-61, 2014. PMCID: PMC3961678.
3) Wang A, Ren J, Wang CP, Hascall VC. Heparin prevents intracellular hyaluronan synthesis and autophagy responses in hyperglycemic dividing mesangial cells and activates synthesis of an extensive extracellular monocyte-adhesive hyaluronan matrix after completing cell division. J Biol Chem. 2014289:9418-29, 2014. PMCID: PMC3979390.
4) Hascall VC, Wang A, Tammi M, Oikari S, Tammi R, Passi A, Vigetti D, Hanson RW, Hart GW. The dynamic metabolism of hyaluronan regulates the cytosolic concentration of UDP-GlcNAc. Matrix Biol. 35:14-7, 2014. PMCID: PMC4039572.
5) Wang A, Midura RJ, Vasanji A, Wang AJ, Hascall VC. Hyperglycemia diverts dividing osteoblastic precursor cells to an adipogenic pathway and induces synthesis of a hyaluronan matrix that is adhesive for monocytes. J. Biol. Chem. 289:11410-20, 2014. PMCID: PMC4036277.
6) Lauer ME, Hascall VC, Green DE, DeAngelis PL, Calabro A. Irreversible heavy chain transfer to chondroitin. J Biol Chem. 289:29171-9, 2014. PMCID: PMC4200270.
7) Vigetti D, Deleonibus S, Moretto P, Bowen T, Fischer JW, Grandoch M, Oberhuber A, Love DC, Hanover JA, Cinquetti R, Karousou E, Viola M, D’Angelo ML, Hascall VC, De Luca G, Passi A. Natural antisense transcript for hyaluronan synthase 2 (HAS2-AS1) induces transcription of HAS2 via protein O-GlcNAcylation. J Biol Chem. 289:28816-26, 2014. PMCID: PMC4200242.
8) Misra S, Ghatak S, Vyas A, O’Brien P, Markwald RR, Khetmalas M, Hascall VC, McCarthy JB, Karamanos NK, Tammi MI, Tammi RH, Prestwitch GD, Padhye S. Isothiocyanate analogs targeting CD44 receptor as an effective strategy against colon cancer. Med Chem Res. ;23:3836-3851, 2014. PMCID: PMC4084864.
9) Coulson-Thomas VJ, Gesteira TF, Hascall V, Kao W. Umbilical cord mesenchymal stem cells suppress host rejection: the role of the glycocalyx. J Biol Chem. 289:23465-81, 2014. PMCID: PMC4156039.
10) Elliott L, Cheng G, Calabro A, Hascall VC, Joo EJ, Li L, Linhardt RJ, Lauer ME. Heavy chain transfer by tumor-necrosis-factor-stimulated-gene-6 to the bikunin proteoglycan. J. Biol. Chem. First published on January 5, 2015, PMC Journal – In Process
11) Misra S, Hascall V, Markwald R, Atanelishvili I, Moreno-Rodriguez, R, Ghatak S. Utilization of GAGs/PGs as carriers for targeted therapy delivery. Int. J. Cell Biol. 2015 (In Press)
Vigetti D, Deleonibus S, Moretto P, Bowen T, Fischer JW, Grandoch M, Oberhuber A, Love DC, Hanover JA, Cinquetti R, Karousou E, Viola M, D’Angelo ML, Hascall VC, De Luca G, Passi A. Natural antisense transcript for hyaluronan synthase 2 (HAS2-AS1) induces transcription of HAS2 via protein O-GlcNAcylation. J Biol Chem. 289:28816-26, 2014. doi: 10.1074/jbc.M114.597401. PMCID: PMC4200242 [Available on 2015/10/17].
Lauer ME, Loftis J, de la Motte C, Hascall VC. Analysis of the heavy-chain modification and TSG-6 activity in pathological hyaluronan matrices. Methods Mol Biol. 1229:543-8, 2015. doi: 10.1007/978-1-4939-1714-3_42. PMCID: PMC4397960 [Available on 2016-01-01].
Lamkin E, Cheng G, Calabro A, Hascall VC, Joo EJ, Li L, Linhardt RJ, Lauer ME. Heavy chain transfer by tumor-necrosis-factor-stimulated-gene-6 to the bikunin proteoglycan. J. Biol. Chem 290:5156-66, 2015. doi: 10.1074/jbc.M114.636258. PMCID: PMC4335249 [Available on 2016-02-20].
Misra S, Hascall VC, Markwald RR, Ghatak S. Interactions between hyaluronan and its receptors (CD44, RHAMM) regulate the activities of inflammation and cancer. Front Immunol. 6:201, 2015. doi: 10.3389/fimmu.2015.00201. eCollection 2015. Review. PMCID: PMC4422082
Wang A, Sankaranarayanan NV, Yanagishita M, Templeton DM, Desai UR, Sugahara K, Wang CP, Hascall VC. Heparin interaction with a receptor on hyperglycemic dividing cells prevents intracellular hyaluronan synthesis and autophagy responses in models of type 1 diabetes. Matrix Biol. 2015 Apr 16. pii: S0945-053X(15)00082-7. doi: 10.1016/j.matbio.2015.04.003. [Epub ahead of print] Review.
Midgley AC, Duggal L, Jenkins R, Hascall V, Steadman R, Phillips AO, Meran S. Hyaluronan regulates bone morphogenetic protein-7-dependent prevention and reversal of myofibroblast phenotype. J Biol Chem. 290:11218-34, 2015. doi: 10.1074/jbc.M114.625939. PMCID: PMC4416830.

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