Fibroblast inflammatory priming determines regenerative versus fibrotic skin repair in reindeer.

Adult mammalian skin wounds heal by forming fibrotic scars. We report that full-thickness injuries of reindeer antler skin (velvet) regenerate, whereas back skin forms fibrotic scar. Single-cell multi-omics reveal that uninjured velvet fibroblasts resemble human fetal fibroblasts, whereas back skin fibroblasts express inflammatory mediators mimicking pro-fibrotic adult human and rodent fibroblasts. Consequently, injury elicits site-specific immune responses: back skin fibroblasts amplify myeloid infiltration and maturation during repair, whereas velvet fibroblasts adopt an immunosuppressive phenotype that restricts leukocyte recruitment and hastens immune resolution. Ectopic transplantation of velvet to scar-forming back skin is initially regenerative, but progressively transitions to a fibrotic phenotype akin to the scarless fetal-to-scar-forming transition reported in humans. Skin regeneration is diminished by intensifying, or enhanced by neutralizing, these pathologic fibroblast-immune interactions. Reindeer represent a powerful comparative model for interrogating divergent wound healing outcomes, and our results nominate decoupling of fibroblast-immune interactions as a promising approach to mitigate scar.

The development of brain pericytes requires expression of the transcription factor nkx3.1 in intermediate precursors.

Brain pericytes are one of the critical cell types that regulate endothelial barrier function and activity, thus ensuring adequate blood flow to the brain. The genetic pathways guiding undifferentiated cells into mature pericytes are not well understood. We show here that pericyte precursor populations from both neural crest and head mesoderm of zebrafish express the transcription factor nkx3.1 develop into brain pericytes. We identify the gene signature of these precursors and show that an nkx3.1-, foxf2a-, and cxcl12b-expressing pericyte precursor population is present around the basilar artery prior to artery formation and pericyte recruitment. The precursors later spread throughout the brain and differentiate to express canonical pericyte markers. Cxcl12b-Cxcr4 signaling is required for pericyte attachment and differentiation. Further, both nkx3.1 and cxcl12b are necessary and sufficient in regulating pericyte number as loss inhibits and gain increases pericyte number. Through genetic experiments, we have defined a precursor population for brain pericytes and identified genes critical for their differentiation.

Comparison of human skin- and nerve-derived Schwann cells reveals many similarities and subtle genomic and functional differences.

Skin is an easily accessible tissue and a rich source of Schwann cells (SCs). Toward potential clinical application of autologous SC therapies, we aim to improve the reliability and specificity of our protocol to obtain SCs from small skin samples. As well, to explore potential functional distinctions between skin-derived SCs (Sk-SCs) and nerve-derived SCs (N-SCs), we used single-cell RNA-sequencing and a series of in vitro and in vivo assays. Our results showed that Sk-SCs expressed typical SC markers. Single-cell sequencing of Sk- and N-SCs revealed an overwhelming overlap in gene expression with the exception of HLA genes which were preferentially up-regulated in Sk-SCs. In vitro, both cell types exhibited similar levels of proliferation, migration, uptake of myelin debris and readily associated with neurites when co-cultured with human iPSC-induced motor neurons. Both exhibited ensheathment of multiple neurites and early phase of myelination, especially in N-SCs. Interestingly, dorsal root ganglion (DRG) neurite outgrowth assay showed substantially more complexed neurite outgrowth in DRGs exposed to Sk-SC conditioned media compared to those from N-SCs. Multiplex ELISA array revealed shared growth factor profiles, but Sk-SCs expressed a higher level of VEGF. Transplantation of Sk- and N-SCs into injured peripheral nerve in nude rats and NOD-SCID mice showed close association of both SCs to regenerating axons. Myelination of rodent axons was observed infrequently by N-SCs, but absent in Sk-SC xenografts. Overall, our results showed that Sk-SCs share near-identical properties to N-SCs but with subtle differences that could potentially enhance their therapeutic utility.

Hair follicle dermal stem cells and skin-derived precursor cells: Exciting tools for endogenous and exogenous therapies.

Understanding the cellular interactions and molecular signals underlying hair follicle (HF) regeneration may have significant implications for restorative therapies for skin disease that diminish hair growth, whilst also serving to provide fundamental insight into the mechanisms underlying adult tissue regeneration. One of the major, yet underappreciated, players in this process is the underlying HF mesenchyme. Here, we provide an overview of a mesenchymal progenitor pool referred to as hair follicle dermal stem cells (hfDSCs), discuss their potential functions within the skin and their relationship to skin-derived precursors (SKPs), and consider unanswered questions about the function of these specialized fibroblasts. We contend that dermal stem cells provide an important reservoir of renewable dermal progenitors that may enable development of novel restorative therapies following hair loss, skin injury or disease.

Imaging Collagen in Scar Tissue: Developments in Second Harmonic Generation Microscopy for Biomedical Applications.

The ability to respond to injury with tissue repair is a fundamental property of all multicellular organisms. The extracellular matrix (ECM), composed of fibrillar collagens as well as a number of other components is dis-regulated during repair in many organs. In many tissues, scaring results when the balance is lost between ECM synthesis and degradation. Investigating what disrupts this balance and what effect this can have on tissue function remains an active area of research. Recent advances in the imaging of fibrillar collagen using second harmonic generation (SHG) imaging have proven useful in enhancing our understanding of the supramolecular changes that occur during scar formation and disease progression. Here, we review the physical properties of SHG, and the current nonlinear optical microscopy imaging (NLOM) systems that are used for SHG imaging. We provide an extensive review of studies that have used SHG in skin, lung, cardiovascular, tendon and ligaments, and eye tissue to understand alterations in fibrillar collagens in scar tissue. Lastly, we review the current methods of image analysis that are used to extract important information about the role of fibrillar collagens in scar formation.

Nonclassical resident macrophages are important determinants in the development of myocardial fibrosis.

Macrophages are increasingly recognized as a potential therapeutic target in myocardial fibrosis via interactions with fibroblasts. We have characterized macrophage depletion and inhibition of nonclassical macrophage migration, in addition to direct interactions between nonclassical macrophages and fibroblasts in angiotensin II (AngII)-mediated, hypertensive myocardial fibrosis. Macrophage depletion was achieved by daily i.v. clodronate liposomes (-1 day to +3 days) during AngII infusion. Cx3cr1(-/-) mice were used to inhibit nonclassical macrophage migration. Macrophage phenotype (F4/80, CD11b, Ly6C) was characterized by immunofluorescence and flow cytometry. Collagen was assessed by Sirius Red/Fast Green. Quantitative real-time RT-PCR was performed for transcript levels. AngII/wild-type (WT) mice displayed significant infiltrate and fibrosis compared with saline/WT, which was virtually ablated by clodronate liposomes independent of hypertension. In vitro data supported M2 macrophages promoting fibroblast differentiation and collagen production. AngII/Cx3cr1(-/-) mice, however, significantly increased macrophage infiltrate and fibrosis relative to AngII/WT. AngII/Cx3cr1(-/-) mice also showed an M1 phenotypic shift relative to WT mice in, which the predominant phenotype was Ly6C(low), CD206(+) (M2). Myocardial IL-1ß was significantly up-regulated, whereas transforming growth factor ß down-regulated with this M1 shift. We demonstrated that infiltrating macrophages are critical to AngII-mediated myocardial fibrosis by preventing the development of fibrosis after liposomal depletion of circulating monocytes. Our findings also suggest that some macrophages, namely M2, may confer a protective myocardial environment that may prevent excessive tissue injury.

Disruption of collagen homeostasis can reverse established age-related myocardial fibrosis.

Heart failure, the leading cause of hospitalization of elderly patients, is correlated with myocardial fibrosis (ie, deposition of excess extracellular matrix proteins such as collagen). A key regulator of collagen homeostasis is lysyl oxidase (LOX), an enzyme responsible for cross-linking collagen fibers. Our objective was to ameliorate age-related myocardial fibrosis by disrupting collagen cross-linking through inhibition of LOX. The nonreversible LOX inhibitor ß-aminopropionitrile (BAPN) was administered by osmotic minipump to 38-week-old C57BL/6J male mice for 2 weeks. Sirius Red staining of myocardial cross sections revealed a reduction in fibrosis, compared with age-matched controls (5.84 ± 0.30% versus 10.17 ± 1.34%) (P < 0.05), to a level similar to that of young mice at 8 weeks (4.9 ± 1.2%). BAPN significantly reduced COL1A1 mRNA, compared with age-matched mice (3.5 ± 0.3-fold versus 15.2 ± 4.9-fold) (P < 0.05), suggesting that LOX is involved in regulation of collagen synthesis. In accord, fibrotic factor mRNA expression was reduced after BAPN. There was also a novel increase in Ly6C expression by resident macrophages. By interrupting collagen cross-linking by LOX, the BAPN treatment reduced myocardial fibrosis. A novel observation is that BAPN treatment modulated the transforming growth factor-ß pathway, collagen synthesis, and the resident macrophage population. This is especially valuable in terms of potential therapeutic targeting of collagen regulation and thereby age-related myocardial fibrosis.

Collagen structural alterations contribute to stiffening of tissue after split-thickness skin grafting.

The gold standard treatment for full thickness injuries of the skin is autologous split-thickness skin grafting. This involves harvesting the epidermis and superficial dermis from healthy skin and transplanting it onto the prepared wound bed. The donor site regenerates spontaneously, but the appendages and cellular components from the dermal layer are excluded from the graft. As a result, the new tissue is inferior; the healed graft site is dry/itchy, has decreased elasticity, increased fragility, and altered sensory function. Because this dermal layer is composed of collagen and other extracellular matrix proteins, the aim was to characterize the changes in the dermal collagen after split thickness grafting that could contribute to a deficit in functionality. This will serve as a baseline for future studies designed to improve skin function using pharmacological or cell-based therapies for skin repair. A xenograft model whereby human split-thickness grafts were implanted into full-thickness defects on immunocompromised (athymic Nu/Nu) mice was used. The grafts were harvested 4 and 8 weeks later. The collagen microstructure was assessed with second harmonic generation with dual-photon microscopy and light polarization analysis. Collagen fiber stiffness and engagement stretch were estimated by fitting the results of biaxial mechanical tensile tests to a histo-mechanical constitutive model. The stiffness of the collagen fibril-proteoglycan complex increased from 682 ± 226 kPa/sr to 1016 ± 324 kPa/sr between 4 and 8 weeks postgrafting. At the microstructural level there were significant decreases in both thickness of collagen fibers (3.60 ± 0.34 µm vs. 2.10 ± 0.27 µm) and waviness ratio (2.04 ± 0.17 vs. 1.43 ± 0.08) of the collagen fibers postgrafting. The decrease of the macroscopic engagement stretch from 1.19 ± 0.11 to 1.09 ± 0.08 over time postgrafting mirrored the decrease in waviness measured at the microscopic level. This suggested that the integrity of the collagen fibers was compromised and contributed to the functional deficit of the skin postgrafting.

Regulation and role of connective tissue growth factor in AngII-induced myocardial fibrosis.

Exposure of rodents to angiotensin II (AngII) is a common model of fibrosis. We have previously shown that cellular infiltration of bone marrow-derived progenitor cells (fibrocytes) occurs before deposition of extracellular matrix and is associated with the production of connective tissue growth factor (CTGF). In the present study, we characterized the role of CTGF in promoting fibrocyte accumulation and regulation after AngII exposure. In animals exposed to AngII using osmotic minipumps (2.0 µg/kg per min), myocardial CTGF mRNA peaked at 6 hours (21-fold; P < 0.01), whereas transforming growth factor-ß (TGF-ß) peaked at 3 days (fivefold; P < 0.05) compared with saline control. Early CTGF expression occurred before fibrocyte migration (1 day) into the myocardium or ECM deposition (3 days). CTGF protein expression was evident by day 3 of AngII exposure and seemed to be localized to resident cells. Isolated cardiomyocytes and microvascular endothelial cells responded to AngII with increased CTGF production (2.1-fold and 2.8-fold, respectively; P < 0.05), which was abolished with the addition of anti-TGF-ß neutralizing antibody. The effect of CTGF on isolated fibrocytes suggested a role in fibrocyte proliferation (twofold; P < 0.05) and collagen production (2.3-fold; P < 0.05). In summary, we provide strong evidence that AngII exposure first resulted in Smad2-dependent production of CTGF by resident cells (6 hours), well before the accumulation of fibrocytes or TGF-ß mRNA up-regulation. In addition, CTGF contributes to fibrocyte proliferation in the myocardium and enhances fibrocyte differentiation into a myofibroblast phenotype responsible for ECM deposition.

Early fibroblast progenitor cell migration to the AngII-exposed myocardium is not CXCL12 or CCL2 dependent as previously thought.

Fibroblast progenitor cells (fibrocytes) are important to the development of myocardial fibrosis and are suggested to migrate to the heart via CXCL12 and chemokine ligand (CCL) 2. We hypothesized that if these chemokines are recruiting fibrocytes, disrupting their signaling will reduce early (3-day) fibrocyte infiltration and, consequently, fibrosis in the myocardium. C57/Bl6 and CCR2(-/-) mice were infused with saline or angiotensin (Ang) II, with or without CXC receptor 4 blockade (AMD3100). Hearts were assessed for chemokine up-regulation, immunofluorescence, and histological features. AngII caused early myocardial up-regulation of CXCL12 and CCL2, which corresponded to significant myocardial infiltration and fibrosis compared with controls. Animals receiving AMD3100 and/or with the genotype CCR2(-/-) failed to demonstrate reductions in infiltrate or fibrosis after 3 days of AngII, and AngII + AMD3100 animals showed exacerbated fibrocyte infiltration and fibrosis compared with AngII alone. CCR2(-/-) mice demonstrated significant reductions in myocardial fibrosis relative to wild type, but this was after 28 days of AngII infusion and was the result of reduced infiltrating cell proliferation. An alternative CCR2 ligand, CCL12, was found to be increasing infiltrating cell proliferation in the heart after AngII infusion, which we confirmed in vitro. In conclusion, early fibrocyte recruitment cannot be inhibited through modulating CXCL12 or CCL2, as previously thought. Ablating CCR2 signaling did confer myocardial fibrosis reductions, but these benefits were not observed until much later and were likely the result of modulated proliferation through ablating the CCL12-CCR2 interaction.

Novel AAV variants with improved tropism for human Schwann cells.

Gene therapies and associated technologies are transforming biomedical research and enabling novel therapeutic options for patients living with debilitating and incurable genetic disorders. The vector system based on recombinant adeno-associated viral vectors (AAVs) has shown great promise in recent clinical trials for genetic diseases of multiple organs, such as the liver and the nervous system. Despite recent successes toward the development of novel bioengineered AAV variants for improved transduction of primary human tissues and cells, vectors that can efficiently transduce human Schwann cells (hSCs) have yet to be identified. Here, we report the application of the functional transduction-RNA selection method in primary hSCs for the development of AAV variants for specific and efficient transgene delivery to hSCs. The two identified capsid variants, Pep2hSC1 and Pep2hSC2, show conserved potency for delivery across various in vitro, in vivo, and ex vivo models of hSCs. These novel AAV capsids will serve as valuable research tools, forming the basis for therapeutic solutions for both SC-related disorders or peripheral nervous system injury.

Single-cell analysis reveals distinct fibroblast plasticity during tenocyte regeneration in zebrafish.

Despite their importance in tissue maintenance and repair, fibroblast diversity and plasticity remain poorly understood. Using single-cell RNA sequencing, we uncover distinct sclerotome-derived fibroblast populations in zebrafish, including progenitor-like perivascular/interstitial fibroblasts, and specialized fibroblasts such as tenocytes. To determine fibroblast plasticity in vivo, we develop a laser-induced tendon ablation and regeneration model. Lineage tracing reveals that laser-ablated tenocytes are quickly regenerated by preexisting fibroblasts. By combining single-cell clonal analysis and live imaging, we demonstrate that perivascular/interstitial fibroblasts actively migrate to the injury site, where they proliferate and give rise to new tenocytes. By contrast, perivascular fibroblast-derived pericytes or specialized fibroblasts, including tenocytes, exhibit no regenerative plasticity. Active Hedgehog (Hh) signaling is required for the proliferation of activated fibroblasts to ensure efficient tenocyte regeneration. Together, our work highlights the functional diversity of fibroblasts and establishes perivascular/interstitial fibroblasts as tenocyte progenitors that promote tendon regeneration in a Hh signaling-dependent manner.

A protocol for single nucleus RNA-seq from frozen skeletal muscle.

Single-cell technologies are a method of choice to obtain vast amounts of cell-specific transcriptional information under physiological and diseased states. Myogenic cells are resistant to single-cell RNA sequencing because of their large, multinucleated nature. Here, we report a novel, reliable, and cost-effective method to analyze frozen human skeletal muscle by single-nucleus RNA sequencing. This method yields all expected cell types for human skeletal muscle and works on tissue frozen for long periods of time and with significant pathological changes. Our method is ideal for studying banked samples with the intention of studying human muscle disease.

Case report: Immune profiling links neutrophil and plasmablast dysregulation to microvascular damage in post-COVID-19 Multisystem Inflammatory Syndrome in Adults (MIS-A).

Despite surviving a SARS-CoV-2 infection, some individuals experience an intense post-infectious Multisystem Inflammatory Syndrome (MIS) of uncertain etiology. Children with this syndrome (MIS-C) can experience a Kawasaki-like disease, but mechanisms in adults (MIS-A) are not clearly defined. Here we utilize a deep phenotyping approach to examine immunologic responses in an individual with MIS-A. Results are contextualized to healthy, convalescent, and acute COVID-19 patients. The findings reveal systemic inflammatory changes involving novel neutrophil and B-cell subsets, autoantibodies, complement, and hypercoagulability that are linked to systemic vascular dysfunction. This deep patient profiling generates new mechanistic insight into this rare clinical entity and provides potential insight into other post-infectious syndromes.

Cystatin C is glucocorticoid responsive, directs recruitment of Trem2+ macrophages, and predicts failure of cancer immunotherapy.

Cystatin C (CyC), a secreted cysteine protease inhibitor, has unclear biological functions. Many patients exhibit elevated plasma CyC levels, particularly during glucocorticoid (GC) treatment. This study links GCs with CyC's systemic regulation by utilizing genome-wide association and structural equation modeling to determine CyC production genetics in the UK Biobank. Both CyC production and a polygenic score (PGS) capturing predisposition to CyC production were associated with increased all-cause and cancer-specific mortality. We found that the GC receptor directly targets CyC, leading to GC-responsive CyC secretion in macrophages and cancer cells. CyC-knockout tumors displayed significantly reduced growth and diminished recruitment of TREM2+ macrophages, which have been connected to cancer immunotherapy failure. Furthermore, the CyC-production PGS predicted checkpoint immunotherapy failure in 685 patients with metastatic cancer from combined clinical trial cohorts. In conclusion, CyC may act as a GC effector pathway via TREM2+ macrophage recruitment and may be a potential target for combination cancer immunotherapy.

Influence of cationic lipid composition on gene silencing properties of lipid nanoparticle formulations of siRNA in antigen-presenting cells.

Lipid nanoparticles (LNPs) are currently the most effective in vivo delivery systems for silencing target genes in hepatocytes employing small interfering RNA. Antigen-presenting cells (APCs) are also potential targets for LNP siRNA. We examined the uptake, intracellular trafficking, and gene silencing potency in primary bone marrow macrophages (bmMΦ) and dendritic cells of siRNA formulated in LNPs containing four different ionizable cationic lipids namely DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA. LNPs containing DLinKC2-DMA were the most potent formulations as determined by their ability to inhibit the production of GAPDH target protein. Also, LNPs containing DLinKC2-DMA were the most potent intracellular delivery agents as indicated by confocal studies of endosomal versus cytoplamic siRNA location using fluorescently labeled siRNA. DLinK-DMA and DLinKC2-DMA formulations exhibited improved gene silencing potencies relative to DLinDMA but were less toxic. In vivo results showed that LNP siRNA systems containing DLinKC2-DMA are effective agents for silencing GAPDH in APCs in the spleen and peritoneal cavity following systemic administration. Gene silencing in APCs was RNAi mediated and the use of larger LNPs resulted in substantially reduced hepatocyte silencing, while similar efficacy was maintained in APCs. These results are discussed with regard to the potential of LNP siRNA formulations to treat immunologically mediated diseases.

Application of an instructive hydrogel accelerates re-epithelialization of xenografted human skin wounds.

Poor quality (eg. excessive scarring) or delayed closure of skin wounds can have profound physical and pyschosocial effects on patients as well as pose an enormous economic burden on the healthcare system. An effective means of improving both the rate and quality of wound healing is needed for all patients suffering from skin injury. Despite wound care being a multi-billion-dollar industry, effective treatments aimed at rapidly restoring the skin barrier function or mitigating the severity of fibrotic scar remain elusive. Previously, a hydrogel conjugated angiopoietin-1 derived peptide (QHREDGS; Q-peptide) was shown to increase keratinocyte migration and improve wound healing in diabetic mice. Here, we evaluated the effect of this Q-Peptide Hydrogel on human skin wound healing using a mouse xenograft model. First, we confirmed that the Q-Peptide Hydrogel promoted the migration of adult human keratinocytes and modulated their cytokine profile in vitro. Next, utilizing our human to mouse split-thickness skin xenograft model, we found improved healing of wounded human epidermis following Q-Peptide Hydrogel treatment. Importantly, Q-Peptide Hydrogel treatment enhanced this wound re-epithelialization via increased keratinocyte migration and survival, rather than a sustained increase in proliferation. Overall, these data provide strong evidence that topical application of QHREDGS peptide-modified hydrogels results in accelerated wound closure that may lead to improved outcomes for patients.

Dexamethasone modulates immature neutrophils and interferon programming in severe COVID-19.

Although critical for host defense, innate immune cells are also pathologic drivers of acute respiratory distress syndrome (ARDS). Innate immune dynamics during Coronavirus Disease 2019 (COVID-19) ARDS, compared to ARDS from other respiratory pathogens, is unclear. Moreover, mechanisms underlying the beneficial effects of dexamethasone during severe COVID-19 remain elusive. Using single-cell RNA sequencing and plasma proteomics, we discovered that, compared to bacterial ARDS, COVID-19 was associated with expansion of distinct neutrophil states characterized by interferon (IFN) and prostaglandin signaling. Dexamethasone during severe COVID-19 affected circulating neutrophils, altered IFNactive neutrophils, downregulated interferon-stimulated genes and activated IL-1R2+ neutrophils. Dexamethasone also expanded immunosuppressive immature neutrophils and remodeled cellular interactions by changing neutrophils from information receivers into information providers. Male patients had higher proportions of IFNactive neutrophils and preferential steroid-induced immature neutrophil expansion, potentially affecting outcomes. Our single-cell atlas (see 'Data availability' section) defines COVID-19-enriched neutrophil states and molecular mechanisms of dexamethasone action to develop targeted immunotherapies for severe COVID-19.

A B1a-natural IgG-neutrophil axis is impaired in viral- and steroid-associated aspergillosis.

The lung naturally resists Aspergillus fumigatus (Af) in healthy individuals, but multiple conditions can disrupt this resistance, leading to lethal invasive infections. Core processes of natural resistance and its breakdown are undefined. We investigated three distinct conditions predisposing to lethal aspergillosis-severe SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection, influenza A viral pneumonia, and systemic corticosteroid use-in human patients and murine models. We found a conserved and essential coupling of innate B1a lymphocytes, Af-binding natural immunoglobulin G antibodies, and lung neutrophils. Failure of this axis concealed Af from neutrophils, allowing rapid fungal invasion and disease. Reconstituting the axis with immunoglobulin therapy reestablished resistance, thus representing a realistic pathway to repurpose currently available therapies. Together, we report a vital host resistance pathway that is responsible for protecting against life-threatening aspergillosis in the context of distinct susceptibilities.

Myocardial fibrosis in response to Angiotensin II is preceded by the recruitment of mesenchymal progenitor cells.

Myocardial fibrosis is characterized by significant extracellular matrix (ECM) deposition. The specific cellular mediators that contribute to the development of fibrosis are not well understood. Using a model of fibrosis with Angiotensin II (AngII) infusion, our aim was to characterize the cellular elements involved in the development of myocardial fibrosis. Male C57Bl/6 and Tie2-GFP mice were given AngII (2.0 mg/kg/min) or saline (control) via mini osmotic pumps for up to 7 days. Hearts were harvested, weighed and processed for analysis. Cellular infiltration and collagen deposition were quantified. Immunostaining was performed for specific markers of leukocytes (CD45, CD11b), myofibroblasts (SMA), endothelial cells (vWF) and hematopoietic progenitor cells (CD133). Bone marrow (BM) origin of infiltrating cells was assessed using GFP(+) chimeric animals. Relative qRT-PCR was performed for pro-fibrotic cytokines (transforming growth factor (TGF)-ß1, CTGF) as well as the chemokine stromal-derived factor (SDF)-1α. Myocardial-infiltrating cells were grown in vitro. AngII exposure resulted in multifocal myocardial cellular infiltration, which preceded extensive ECM deposition. A limited number of myocardial-infiltrating cells were positive for leukocyte markers but were significantly positive for myofibroblast (SMA) and endothelial cell (vWF) markers. However, using Tie2-GFP mice, where endothelial cells are GFP(+), myocardial-infiltrating cells were not GFP(+). Transcript levels for SDF-1α were significantly elevated at 1 day of AngII exposure suggesting that hematopoietic progenitor cells may be recruited. This was confirmed by positive CD133 staining of infiltrating cells and evident GFP(+) cellular infiltration when exposing GFP(+) BM chimeras to AngII. Furthermore, a significant number of CD133(+)/SMA(+) cells were grown in vitro from the myocardium of AngII-exposed animals (P<0.01). Myocardial ECM deposition is preceded by the infiltration of the myocardium with hematopoietic cells that express mesenchymal markers. These data suggest that mesenchymal progenitor cells are recruited, and may have a primary role, in the initiation of myocardial fibrosis.