Granulation tissues contraction was significantly delayed in the KO mice compared to WT mice on day 3, 4, and 5 (Fig. tissue contraction was diminished by ALK5 inhibition, but not JNK. AngII, promotes granulation tissue contraction through AT1-receptor and downstream canonical TGF signaling pathway; ALK5. Further understanding the pathogenesis of HSc as an integrated signaling mechanism could improve our approach to establishing effective therapeutic interventions. Introduction Disability due to hypertrophic scar contraction (HSc) following burn wounds results in approximately $80.2 billion in lost wages worldwide annually [1]. The additional impact of medical expenses, social costs, and emotional trauma due to disfigurement is immeasurable. HSc leads to contractures which are inelastic, thickened scars that fail to regress [2, 3]. These fixed lesions cause pain, deformity, profound itching, and severe disability of joints [3C5]. Contractures as little as a 10% reduction in joint motion is clinically significant [6]. Current anti-HSc therapies are ineffective [7]. Thus, there remains an urgent need to understand the pathogenesis of HSc and identify targets to prevent this disabling process. Over the last half century, it has been established that HSc is mediated by myofibroblast and fibroblast contractility and migration. While contractility and migration are known to be caused by intracellular focal adhesion complex formation, cytoskeletal protein activation, and upregulation included but not limited to -smooth muscle actin (ASMA) [8], vimentin [7], non-muscle myosin (NM IIA) [9], and the regulatory proteins RhoA and Rho Kinase (ROCK) [9], it is unknown, which extracellular soluble substances activate these pathways. Angiotensin II (AngII) is one soluble mediator that has been implicated in stimulating pro-fibrotic processes in heart, liver, kidney, and the lung [10C13]. There is also accumulating evidence that AngII is a key effector in promoting dermal wound healing and fibrosis [14C17]. AngII signals through two receptors: angiotensin type 1 receptor (AT1-receptor) and angiotensin type 2 receptor (AT2-receptor). It has been hypothesized that the balance between AT1-receptor vs. AT2-receptor activation determines healing versus fibrosis, but this hypothesis has not been conclusively tested [18, 19]. AngII has been linked to upregulation of TGF PFK15 production and activation of the TGF signaling pathways; canonical and non-canonical. In the canonical pathway, TGF binds to receptor activin receptor like kinase 5 (ALK5) which activates the phosphorylation of Smad2/3. Activated Smad2/3 proteins recruit Smad4, and cause nuclear translocation of the Smad2/4 or Smad3/4 for activation of pro-contractile mRNA transcription [20]. The TGF non-canonical signaling pathway involves activation of mitogen activated protein kinase (MAPK) pathways including, extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), and p38 [21]. Here we investigate the AngII signaling mechanism involved in dermal HSc using a combination of experiments on human tissue, human and murine cell lines, and murine granulation tissue contraction PFK15 models. Materials and Methods Animals All protocols were approved by Duke University Medical Center Institutional Animal Care and Use Committee. Two strains of 10C12 week -old mice weighing 18C20g were used; (1) C57BL/6J from Jackson Laboratory, PFK15 and (2) KO mice from Thomas Coffman, MD, Duke. Dermal excisional wounding C57BL/6J mice were randomly divided into control (n=12), Losartan (Sigma Aldrich, St. Louis, MI) treated group (n=12) fed 50mg/kg/day Losartan by 0.4 ml saline gavage, and AngII (Calbiochem, San Diego, CA) treated group (n=36) implanted with sub-dermal osmotic pumps (ALZET, Cupertino, CA) to administer 2200ng/kg/min AngII dissolved in PBS. AngII group had three subgroups (n=8) each administered either ALK-inhibitor on day 3 (10mg/kg of 5mg/0.25ml SB431542 (Tocris Bioscience, Bristol, UK) in DMSO by CREB3L4 intraperitoneal (IP) injection), JNK-inhibitor for 14 days (16mg/kg/day of SP600125 (Sigma-Aldrich) in 45% w/v 2-hydroxypropyl-B cyclodextrin IP), or equivalent amounts of PBS. All PFK15 mice, including KO (n=5), were halothane-anaesthetized and 8-mm full-thickness excisional wounds created on the dorsum. The wound area was measured using gravitational planimetry and expressed as a percentage of original wound size. On days 3, 7, and 14, animals (n=5 per time point per group) were euthanized, and the wound bed was excised with a 5mm margin of unwounded skin for histological and immunohistochemical (IHC) staining. Histology To determine epidermal thickness, collagen deposition, PFK15 and mast cell infiltration, histological analysis was performed on excised samples by staining with hematoxylin and eosin, Massons Trichrome, and Toluidine blue respectively. Immunohistochemistry Samples of human studies were obtained after.
