PDGFRα signaling drives adipose tissue fibrosis by targeting progenitor cell plasticity.

Fibrosis is a common disease process in which profibrotic cells disturb organ function by secreting disorganized extracellular matrix (ECM). Adipose tissue fibrosis occurs during obesity and is associated with metabolic dysfunction, but how profibrotic cells originate is still being elucidated. Here, we use a developmental model to investigate perivascular cells in white adipose tissue (WAT) and their potential to cause organ fibrosis. We show that a Nestin-Cre transgene targets perivascular cells (adventitial cells and pericyte-like cells) in WAT, and Nestin-GFP specifically labels pericyte-like cells. Activation of PDGFRα signaling in perivascular cells causes them to transition into ECM-synthesizing profibrotic cells. Before this transition occurs, PDGFRα signaling up-regulates mTOR signaling and ribosome biogenesis pathways and perturbs the expression of a network of epigenetically imprinted genes that have been implicated in cell growth and tissue homeostasis. Isolated Nestin-GFP(+) cells differentiate into adipocytes ex vivo and form WAT when transplanted into recipient mice. However, PDGFRα signaling opposes adipogenesis and generates profibrotic cells instead, which leads to fibrotic WAT in transplant experiments. These results identify perivascular cells as fibro/adipogenic progenitors in WAT and show that PDGFRα targets progenitor cell plasticity as a profibrotic mechanism.

Retinal laser burn-induced neuropathy leads to substance P-dependent loss of ocular immune privilege.

Inflammation in the eye is tightly regulated by multiple mechanisms that together contribute to ocular immune privilege. Many studies have shown that it is very difficult to abrogate the immune privileged mechanism called anterior chamber-associated immune deviation (ACAID). Previously, we showed that retinal laser burn (RLB) to one eye abrogated immune privilege (ACAID) bilaterally for an extended period of time. In an effort to explain the inflammation in the nonburned eye, we postulated that neuronal signals initiated inflammation in the contralateral eye. In this study, we test the role of substance P, a neuroinflamatory peptide, in RLB-induced loss of ACAID. Histological examination of the retina with and without RLB revealed an increase of the substance P-inducible neurokinin 1 receptor (NK1-R) in the retina of first, the burned eye, and then the contralateral eye. Specific antagonists for NK1-R, given locally with Ag within 24 h, but not 3, 5, or 7 d post-RLB treatment, prevented the bilateral loss of ACAID. Substance P knockout (KO) mice retained their ability to develop ACAID post-RLB. These data support the postulate that substance P transmits early inflammatory signals from the RLB eye to the contralateral eye to induce changes to ocular immune privilege and has a central role in the bilateral loss of ACAID. The possibility is raised that blocking of the substance P pathway with NK1-R antagonists postocular trauma may prevent unwanted and perhaps extended consequences of trauma-induced inflammation in the eye.

Growth factor regulation of corneal keratocyte differentiation and migration in compressed collagen matrices.

PURPOSE: To evaluate a novel 3D culture model of the corneal stroma and apply it to investigate how key wound-healing growth factors regulate the mechanics of corneal keratocyte migration. METHODS: Rabbit corneal keratocytes were seeded within collagen matrices that were compacted using external compression. Six-millimeter-diameter buttons were then incubated in media supplemented with 10% FBS, TGFbeta1, TGFbeta2, platelet-derived growth factor (PDGF), or no growth factor (control). After 1, 3, or 7 days, matrices were labeled with phalloidin and a nucleic acid dye, and were imaged using laser confocal microscopy. To study cell migration, buttons were nested within acellular uncompressed outer collagen matrices before growth factor stimulation. RESULTS: Corneal keratocytes in basal media within compressed matrices had a broad, convoluted cell body and thin dendritic processes. In contrast, cells in 10% FBS developed a bipolar fibroblastic morphology. Treatment with TGFbeta induced the formation of stress fibers expressing alpha-smooth muscle actin, suggesting myofibroblast transformation. PDGF induced keratocyte elongation without inducing stress fiber formation. Both 10% FBS and PDGF stimulated significant keratocyte migration through the uncompressed outer matrix, but 10% FBS produced more cell-induced collagen matrix reorganization. TGFbeta induced the smallest increase in migration and the greatest matrix reorganization. CONCLUSIONS: Corneal keratocytes are able to differentiate normally and respond to growth factors within compressed collagen matrices, which provide a high-stiffness, 3D environment, similar to native stromal tissue. In addition, nesting these matrices provides a unique platform for investigating the mechanics of keratocyte migration after exposure to specific wound-healing cytokines.

Human corneal fibrosis: an in vitro model.

PURPOSE: Corneal injury may ultimately lead to a scar by way of corneal fibrosis, which is characterized by the presence of myofibroblasts and improper deposition of extracellular matrix (ECM) components. TGF-beta1 is known to stimulate overproduction and deposition of ECM components. Previously, an in vitro three-dimensional (3-D) model of a corneal stroma was developed by using primary human corneal fibroblasts (HCFs) stimulated with stable vitamin C (VitC). This model mimics corneal development. The authors postulate that with the addition of TGF-beta1, a 3-D corneal scar model can be generated. METHODS: HCFs were grown in four media conditions for 4 or 8 weeks: VitC only; VitC+TGF-beta1 for the entire time; VitC+TGF-beta1 for 1 week, then VitC only for 3 or 7 weeks; and VitC for 4 weeks, then VitC+TGF-beta1 for 4 weeks. Cultures were analyzed with TEM and indirect immunofluorescence. RESULTS: Compared with the control, addition of TGF-beta1 increased construct thickness significantly, with maximum increase in constructs with TGF-beta1 present for the entire time-2.1- to 3.2-fold at 4 and 8 weeks, respectively. In all TGF-beta-treated cultures, cells became long and flat, numerous filamentous cells were seen, collagen levels increased, and long collagen fibrils were visible. Smooth muscle actin, cellular fibronectin, and type III collagen expression all appeared to increase. Cultures between weeks 4 and 8 showed minimal differences. CONCLUSIONS: Human corneal fibroblasts stimulated by VitC and TGF-beta1 appear to generate a model that resembles processes observed in human corneal fibrosis. This model should be useful in examining matrix deposition and assembly in a wound-healing situation.

An experimental model for assessing fibroblast migration in 3-D collagen matrices.

The purpose of this study was to develop and test a novel culture model for studying fibroblast migration in 3-D collagen matrices. Human corneal fibroblasts were seeded within dense, randomly oriented compressed collagen matrices. A 6 mm diameter button of this cell-seeded matrix was placed in the middle of an acellular, less dense outer collagen matrix. These constructs were cultured for 1, 3, 5 or 7 days in serum-free media, 10% fetal bovine serum (FBS), or 50 ng/ml PDGF. Constructs were then fixed and labeled with AlexaFluor 546 phalloidin (for f-actin) and TOTO-3 (for nuclei). Cell-matrix interactions were assessed using a combination of fluorescent and reflected light confocal imaging. Human corneal fibroblasts in serum-free media showed minimal migration into the outer (non-compressed) matrix. In contrast, culture in serum or PDGF stimulated cell migration. Cell-induced collagen matrix reorganization in the outer matrix could be directly visualized using reflected light imaging, and was highest following culture in 10% FBS. Cellular contraction in 10% FBS often led to alignment of cells parallel to the outer edge of the inner matrix, similar to the pattern observed during corneal wound healing following incisional surgery. Overall, this 3-D model allows the effects of different culture conditions on cell migration and matrix remodeling to be assessed simultaneously. In addition, the design allows for ECM density, geometry and mechanical constraints to be varied in a controlled fashion. These initial results demonstrate differences in cell and matrix patterning during migration in response to serum and PDGF.

Matrix stiffness and serum concentration effects matrix remodelling and ECM regulatory genes of human bone marrow stem cells.

The effects of mechanical stimulation of cell-seeded collagen constructs on cell orientation, intracellular signalling and molecular responses have been widely reported. In this study we investigated in vitro the contractile responses of human bone marrow stem cells (HBMSCs) to increasing collagen gel substrate stiffness and their effect on extracellular matrix (ECM) regulatory genes. Human dermal fibroblasts (HDFs) were used as controls. Cells were cultured in 10% and 20% FCS and embedded in collagen constructs at a density of 1 million cells/ml collagen. Matrix stiffness was achieved by subjecting the constructs to three different strain regimes (0%, 5% and 10%), using a computer-driven tensional culture force monitor (t-CFM) capable of uniaxial loading. The contraction forces generated by the cells were quantified over 24 h. Molecular outputs were quantified using RT-PCR. HBMSCs significantly increased force generation to increasing serum concentration (i.e 10% to 20%). 10% FCS concentration significantly reduced contraction as pre-strain stiffness was increased in HBMSCs and HDFs (0% > 5% > 10%). However, at 20% FCS HBMSCs generated similar peak force contraction at 24 h to 5% and 10% pre-strain (0% = 5% = 10%). The ECM regulatory gene for MMP2 showed upregulation at 5% pre-strain, but a 50% downregulation when pre-strain was increased to 10%. MMP9 was upregulated at 5% pre-strain and further upregulated at 10% pre-strain. In designing tissue-engineering solutions, predictable responses of cells, embedded within bio-artificial matrices, to external mechanical forces are critical. To take into account the increasing stiffness of the matrix as increasing ECM is deposited, it would be necessary to take mechanical stimulation into account to determine predictable cellular responses.

Regulation of corneal fibroblast morphology and collagen reorganization by extracellular matrix mechanical properties.

PURPOSE: To investigate how extracellular matrix mechanical properties influence cell and matrix patterning in three-dimensional culture. METHODS: Human corneal fibroblasts were seeded within 30 x 10 mm collagen matrices that were unconstrained (UN), fully constrained (CO) along the long axis by attaching the construct to two immobilized plastic bars, or partially constrained (PC) by allowing linear elastic displacement of one bar. After 24 hours, constructs were labeled with phalloidin and were imaged using fluorescent and reflected light (for collagen) confocal microscopy. Cell morphology and local collagen fibril density and alignment were measured using digital image processing. RESULTS: Corneal fibroblasts in UN matrices were less elongated (UN < PC < CO; P < 0.05) than those in constrained matrices. Cells were aligned parallel to the long axis in the anisotropic region of constrained matrices but were randomly aligned in unconstrained (isotropic) matrices (UN < PC = CO; P < 0.05). Both the local collagen density and the degree of cell/collagen coalignment were higher in constrained matrices (UN < PC < CO; P < 0.05). In regions of higher cell density, additional bands of aligned collagen were often observed between individual cells. CONCLUSIONS: These data suggest that cell spreading, alignment, and contractile force generation are directly influenced by the mechanical properties of the surrounding extracellular matrix (ECM). Corneal fibroblasts generally align and compact collagen parallel to the axis of greatest ECM stiffness. Mechanical cross-talk between adjacent cells leads to enhancement of matrix reorganization, and results in additional, more complex matrix patterning.