Projects

Hyaluronan Matrices in Vascular Pathologies

Project 1: Glycosaminoglycans in Diabetic Vascular Pathology

We have shown that glomerular mesangial cells that divide in diabetic levels of glucose initiate hyaluronan synthesis in intracellular compartments (endoplasmic reticulum, Golgi, transport vesicles). This causes a novel autophagy that extrudes the hyaluronan into the extracellular matrix. Our new data show that this process also occurs in kidney vascular endothelial cells and associated connective tissue cells. Our goals are to determine the mechanisms involved and how heparin, which has been used in clinical trials with diabetic patients, alters this process.

Project 1 Update: Last year’s 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.  Continued …

Personnel

Project 2: Hyaluronan as a Regulator of Inflammation and Fibrosis

We have shown that the pathological hyaluronan matrix is synthesized at the junction between the dermal and adipose layers adjacent to postcapillary venules early during wound healing, the area where the influx of neutrophils occurs and where myofibroblasts appear. Our goals are to determine how the hyaluronan matrix synthesized by the vascular endothelial cells regulates neutrophil recruitment and regulates myofibroblast transformation through interactions between the hyaluronan receptor CD44 and transforming growth factor-.

Project 2 Update: Project 2 continues to contribute data to elucidate the functional involvement of hyaluronan (HA) in wound healing and cutaneous responses to injury.  The cornerstone of these studies is the Has1/Has3 double-knockout mice (Has1/Has3 null) that we created, mice in which only one functional HA-synthetic enzyme (Has2) remains. Continued…

Personnel

  • Judith Mack, PhD (Co-Investigator)
  • (216) 445-6676

Project 3: Platelet Production and Modification of Inflammatory Hyaluronan Fragments in Colitis

We have shown: 1) that platelets contain hyaluronan and have hyaluronidase-2 on their surfaces, and 2) that platelets bind to the pathological hyaluronan matrix and cleave the hyaluronan into fragments that are released and considered to be ‘endogenous danger signals’ for upregulating innate immune responses. Our goal is to determine the cascade of events that bring platelets to vascular endothelial cell surfaces to initiate the fragmentation of hyaluronan and its exacerbation of inflammation in a mouse model of colitis and how deletion of hyaluronan synthase-3 and hyaluronidase-2 in platelets will alter the inflammatory responses.

Update to Project 3:  

AIM 1. This year we have completed and published the results of most of the proposed experiments within this aim (Blood 2014, ref. #4 below). We reported: 1) a role for Hyaluronidase 2 (HYAL2) and necessity for activation that allows platelets to cleave the HA -rich pathologic HA matrix; and 2) the discovery that patients with Inflammatory Bowel Disease (IBD) have greatly decreased levels of platelet HYAL2 (Blood 2014).  Continued…

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Project 4: Hyaluronan in the Lung

We have shown that the pathological hyaluronan matrix is central for the aberrant vascular angiogenesis that occurs in patients with idiopathic pulmonary arterial hypertension, a fatal disease. Abnormal proliferation of pulmonary smooth muscle cells is a major component of the remodeling process that is driving this disease. Our goal is to determine the mechanisms involved in the synthesis of the pathological hyaluronan matrix by pulmonary smooth muscle cells isolated from lungs of patients compared to those isolated from normal lungs.

Project 4 Update: The overall goal of our proposed studies is to understand the mechanisms for the increased production of HA by PASMCs through the study of the effects of NO on the abnormal HA matrix in IPAH. The innovative approach in this proposal is enhanced exponentially by sharing several common themes with other components in the Program Project.  Continued…

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Project 5: Versican Proteolysis and Regulation of Vascular Smooth Muscle

The dynamic pericellular matrix (glycocalyx) of vascular smooth muscle cells contains the proteoglycan versican bound to hyaluronan. We have shown that the normal steady state catabolism of this matrix requires proteolytic removal of the glycosaminoglycan domain of versican by select metalloproteases of the ADAMTS family, and that the absence of this cleavage in dermal fibroblasts expands the pericellular matrix, upregulates transforming growth factor- and promotes myofibroblast transformation. Our goal is to use transgenic mouse models for the key proteases and cell biological approaches to determine how the abnormal accumulation of the excessive versican-hyaluronan matrix contributes to cardiac, valvular and arterial anomalies.

Project 5 Update: 

AIM 1: We are revising a manuscript (for Cell Reports) that shows how ADAMTS9 regulates the growth of arteries. Specifically, we show, using a new gene-trap mouse allele, that ADAMTS9 is essential for matrix remodeling in the umbilical cord that is a prerequisite for differentiation and proper orientation of vascular smooth muscle cells. Since null mice with a conventionally targeted Adamts9 allele die before cardiovascular development, we developed a floxed Adamts9 allele, with which conditional developmental and inducible post-natal Adamts9 deletion in VSMCs will be achieved. Combinatorial ADAMTS-deficient mice (i.e., Adamts1/Adamts4Adamts5 + Adamts9) will be made to determine their cooperative roles in the vascular wall.  Continued…

Personnel

  • Suneel Apte, MBBS/DPhil (Project Leader)
  • (216) 445-3278