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. These mice exhibit an exaggerated inflammatory and angiogenic response following skin injury, and the hyperinflammatory response appears to lead to excessive fibrosis as indicated by a higher number of fibroblasts expressing alpha-smooth muscle actin (-SMA) in Has1/Has3 null wounds. Efforts during this period have been aimed at trying to understand mechanisms that drive this hyperinflammatory and profibrotic phenotype following injury.

AIM 1: Determine mechanisms by which leukocytes interact with endothelial cells and extravasate more quickly from small cutaneous blood vessels.

We tried for over 6 months to isolate primary endothelial cells (EC) from the skin of C57BL/6 wildtype and Has1/Has3 mice, a procedure not well-documented in the literature, and finally succeeded in isolating pure cultures of CD31-positive primary ECs. However, due to the technical difficulty of obtaining adequate amounts of primary murine ECs for further experimentation, we pursued an alternative approach, which was to obtain a well-characterized line of immortalized human microvascular endothelial cells (HIMEC) from Dr. Swerlick at the CDC, which can be grown in large amounts and remains stable for many passages. So far we have confirmed that the ECs express negligible amounts of Has1, Has2, and Has3 and do not synthesize detectable HA under resting conditions. This contrasts with our in vivo observation in wounded skin that the endothelium of small vessels is associated with an HA coat, although the exact microanatomical location of HA (apical or basal surface) has yet to be determined. TNF stimulation fails to increase HA expression in the cultured HIMECs, implying that our cultured EC system is missing one or more crucial elements associated with the wound environment, most likely a combination of multiple cytokines or paracrine factors released from nearby pericytes or smooth muscle cells. A third possibility (however unlikely) is that ECs may never make their own HA, but instead collect it from the surrounding environment through enhanced binding. To help us place these findings into physiological context, we participated in writing a comprehensive review on the role of glycosaminoglycans and HA in wound healing, Ghatak S, Markwald RR, Hascall VC, Maytin EV, Mack JA, Atanelishvili I, Rodriguez RM, Misra S, Roles of proteoglycans and glycosaminoglycans in wound healing and fibrosis, Int J Cell Biol (2015), which is currently in press. Going forward, our approach will be to systematically add each of the possible elements to the EC culture system until conditions that stimulate HA production in the cultures are ascertained.

AIM 2: Determine how fibroblasts in Has1/Has3 null skin become activated to exhibit increased matrix deposition in wounds.

We have made two critical observations here. First, in Has1/Has3 null skin wounds in vivo we found that expression of the profibrotic cytokine TGF1 is significantly elevated and prolonged, correlating with the influx of leukocytes from cutaneous blood vessels in these mice. Second, Has1/Has3 knockout skin fibroblasts in culture exhibit a unique and interesting phenotype that features the following aspects: (i) the cells synthesize more HA than do normal fibroblasts, due to a significant elevation in Has2 expression; (ii) they secrete a relatively larger HA-containing pericellular coat; (iii) they are more resistant to apoptotic death after environmental stress such as serum starvation or exposure to UVB. These findings were published in Wang Y, Lauer ME, Anand S, Mack JA, Maytin EV, Hyaluronan synthase 2 protects skin fibroblasts against apoptosis induced by environmental stress, J Biol Chem. 289(46): 32253-65 (2014), (PMCID: PMC4231699). The first and second result, taken together, may allow us to unify some previous observations and explain how an accelerated inflammatory cell influx might be linked to accelerated production of the collagen-containing matrix in the healing dermis in the Has1/Has2 null mice. An enlarged HA coat on fibroblasts may increase the number of fibroblasts (via inhibition of apoptosis), and also augment TGF1 receptor signaling that drives fibroblast-to myofibroblast conversion, collagen synthesis, and fibroblast contractility.

The most important point is that we are reaching out to other members of the PEG and will build upon an expanding set of collaborators to help us answer the questions and achieve the goals of the project.

AIM 1: Collaboration with Dr. Justin Lathia (Stem Cell Biology, Cleveland Clinic), to study leukocyte transmigration from inflamed vessels in Has1/Has3 mice using intravital imaging techniques.

Collaboration with Dr. Suneel Apte (Cleveland Clinic PEG Project #5), to examine the coordinated expression of vesican and HA in the glycocalyx of endothelial cells and fibroblasts from Has1/Has3 deficient mice.

AIM 2: Collaboration with Dr. Gerald Hart (Cardio PEG), to investigate possible links between elevated levels of Has2 and intracellular HA, apoptosis resistance, UDP-sugar cycling, and O-GlcNAcylation of mitochondrial proteins.

Collaboration with Dr. Mark Lauer (Cleveland Clinic PEG) to measure and describe HA responses in the skin, including changes in TSG-6 mediated cross-linking in healing wounds.

1) Wang Y, Lauer ME, Anand S, Mack JA, Maytin EV. Hyaluronan synthase 2 protects skin fibroblasts against apoptosis induced by environmental stress. J Biol Chem. 2014 Nov 14; 289(46):32253-65. PMCID: PMC4231699

2) Ghatak S, Markwald RR, Hascall VC, Maytin EV, Mack JA, Atanelishvili I, Rodriguez RM, Misra S. Roles of proteoglycans and glycosaminoglycans in wound healing and fibrosis. Int J Cell Biol (2015 accepted).

 
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