Differentiated Smooth Muscle Cells Generate a Subpopulation of Resident Vascular Progenitor Cells in the Adventitia Regulated by Klf4.

RATIONALE: The vascular adventitia is a complex layer of the vessel wall consisting of vasa vasorum microvessels, nerves, fibroblasts, immune cells, and resident progenitor cells. Adventitial progenitors express the stem cell markers, Sca1 and CD34 (adventitial sca1-positive progenitor cells [AdvSca1]), have the potential to differentiate in vitro into multiple lineages, and potentially contribute to intimal lesions in vivo. OBJECTIVE: Although emerging data support the existence of AdvSca1 cells, the goal of this study was to determine their origin, degree of multipotency and heterogeneity, and contribution to vessel remodeling. METHODS AND RESULTS: Using 2 in vivo fate-mapping approaches combined with a smooth muscle cell (SMC) epigenetic lineage mark, we report that a subpopulation of AdvSca1 cells is generated in situ from differentiated SMCs. Our data establish that the vascular adventitia contains phenotypically distinct subpopulations of progenitor cells expressing SMC, myeloid, and hematopoietic progenitor-like properties and that differentiated SMCs are a source to varying degrees of each subpopulation. SMC-derived AdvSca1 cells exhibit a multipotent phenotype capable of differentiating in vivo into mature SMCs, resident macrophages, and endothelial-like cells. After vascular injury, SMC-derived AdvSca1 cells expand in number and are major contributors to adventitial remodeling. Induction of the transcription factor Klf4 in differentiated SMCs is essential for SMC reprogramming in vivo, whereas in vitro approaches demonstrate that Klf4 is essential for the maintenance of the AdvSca1 progenitor phenotype. CONCLUSIONS: We propose that generation of resident vascular progenitor cells from differentiated SMCs is a normal physiological process that contributes to the vascular stem cell pool and plays important roles in arterial homeostasis and disease.

Vascular smooth muscle cell-derived transforming growth factor-ß promotes maturation of activated, neointima lesion-like macrophages.

OBJECTIVE: To define the contribution of vascular smooth muscle cell (SMC)-derived factors to macrophage phenotypic modulation in the setting of vascular injury. APPROACH AND RESULTS: By flow cytometry, macrophages (M4) were the predominant myeloid cell type recruited to wire-injured femoral arteries, in mouse, compared with neutrophils or eosinophils. Recruited macrophages from injured vessels exhibited a distinct expression profile relative to circulating mononuclear cells (peripheral blood monocytes; increased: interleukin-6, interleukin-10, interleukin-12b, CC chemokine receptor [CCR]3, CCR7, tumor necrosis factor-α, inducible nitric oxide synthase, arginase 1; decreased: interleukin-12a, matrix metalloproteinase [MMP]9). This phenotype was recapitulated in vitro by maturing rat bone marrow cells in the presence of macrophage-colony stimulating factor and 20% conditioned media from cultured rat SMC (sMϕ) compared with maturation in macrophage-colony stimulating factor alone (M0). Recombinant transforming growth factor (TGF)-ß1 recapitulated the effect of SMC conditioned media. Macrophage maturation studies performed in the presence of a pan-TGF-ß neutralizing antibody, a TGF-ß receptor inhibitor, or conditioned media from TGF-ß-depleted SMCs confirmed that the SMC-derived factor responsible for macrophage activation was TGF-ß. Finally, the effect of SMC-mediated macrophage activation on SMC biology was assessed. SMCs cocultured with sMϕ exhibited increased rates of proliferation relative to SMCs cultured alone or with M0 macrophages. CONCLUSIONS: SMC-derived TGF-ß modulates the phenotype of maturing macrophages in vitro, recapitulating the phenotype found in vascular lesions in vivo. SMC-modulated macrophages induce SMC activation to a greater extent than control macrophages.

Activation of the retinoid X receptor modulates angiotensin II-induced smooth muscle gene expression and inflammation in vascular smooth muscle cells.

The retinoid X receptor (RXR) partners with numerous nuclear receptors, such as the peroxisome proliferator activated receptor (PPAR) family, liver X receptors (LXRs), and farnesoid X receptor (FXR). Although each heterodimer can be activated by specific ligands, a subset of these receptors, defined as permissive nuclear receptors, can also be activated by RXR agonists known as rexinoids. Many individual RXR heterodimers have beneficial effects in vascular smooth muscle cells (SMCs). Because rexinoids can potently activate multiple RXR pathways, we hypothesized that treating SMCs with rexinoids would more effectively reverse the pathophysiologic effects of angiotensin II than an individual heterodimer agonist. Cultured rat aortic SMCs were pretreated with either an RXR agonist (bexarotene or 9-cis retinoic acid) or vehicle (dimethylsulfoxide) for 24 hours before stimulation with angiotensin II. Compared with dimethylsulfoxide, bexarotene blocked angiotensin II-induced SM contractile gene induction (calponin and smooth muscle-α-actin) and protein synthesis ([(3)H]leucine incorporation). Bexarotene also decreased angiotensin II-mediated inflammation, as measured by decreased expression of monocyte chemoattractant protein-1 (MCP-1). Activation of p38 mitogen-activated protein (MAP) kinase but not extracellular signal-related kinase (ERK) or protein kinase B (Akt) was also blunted by bexarotene. We compared bexarotene to five agonists of nuclear receptors (PPARα, PPARγ, PPARδ, LXR, and FXR). Bexarotene had a greater effect on calponin reduction, MCP-1 inhibition, and p38 MAP kinase inhibition than any individual agonist. PPARγ knockout cells demonstrated blunted responses to bexarotene, indicating that PPARγ is necessary for the effects of bexarotene. These data demonstrate that RXR is a potent modulator of angiotensin II-mediated responses in the vasculature, partially through inhibition of p38.

Serum response factor regulates expression of phosphatase and tensin homolog through a microRNA network in vascular smooth muscle cells.

OBJECTIVE: Serum response factor (SRF) is a critical transcription factor in smooth muscle cells (SMCs) controlling differentiation and proliferation. Our previous work demonstrated that depleting SRF in cultured SMCs decreased expression of SMC markers but increased proliferation and inflammatory mediators. A similar phenotype has been observed in SMCs silenced for phosphatase and tensin homolog (PTEN), suggesting that SRF and PTEN may lie on a common pathway. Our goal was to determine the effect of SRF depletion on PTEN levels and define mechanisms mediating this effect. METHODS AND RESULTS: In SRF-silenced SMCs, PTEN protein levels but not mRNA levels were decreased, suggesting posttranscriptional regulation. Reintroduction of PTEN into SRF-depleted SMCs reversed increases in proliferation and cytokine/chemokine production but had no effect on SMC marker expression. SRF-depleted cells showed decreased levels of microRNA (miR)-143 and increased miR-21, which was sufficient to suppress PTEN. Increased miR-21 expression was dependent on induction of Fos related antigen (FRA)-1, which is a direct target of miR-143. Introducing miR-143 into SRF-depleted SMCs reduced FRA-1 expression and miR-21 levels and restored PTEN expression. CONCLUSIONS: SRF regulates PTEN expression in SMCs through a miR network involving miR-143, targeting FRA-1, which regulates miR-21. Cross-talk between SRF and PTEN likely represents a critical axis in phenotypic remodeling of SMCs.

SDF-1α induction in mature smooth muscle cells by inactivation of PTEN is a critical mediator of exacerbated injury-induced neointima formation.

OBJECTIVE: PTEN inactivation selectively in smooth muscle cells (SMC) initiates multiple downstream events driving neointima formation, including SMC cytokine/chemokine production, in particular stromal cell-derived factor-1α (SDF-1α). We investigated the effects of SDF-1α on resident SMC and bone marrow-derived cells and in mediating neointima formation. METHODS AND RESULTS: Inducible, SMC-specific PTEN knockout mice (PTEN iKO) were bred to floxed-stop ROSA26-ß-galactosidase (ßGal) mice to fate-map mature SMC in response to injury; mice received wild-type green fluorescent protein-labeled bone marrow to track recruitment. Following wire-induced femoral artery injury, ßGal(+) SMC accumulated in the intima and adventitia. Compared with wild-type, PTEN iKO mice exhibited massive neointima formation, increased replicating intimal and medial ßGal(+)SMC, and enhanced vascular recruitment of bone marrow cells following injury. Inhibiting SDF-1α blocked these events and reversed enhanced neointima formation observed in PTEN iKO mice. Most recruited green fluorescent protein(+) cells stained positive for macrophage markers but not SMC markers. SMC-macrophage interactions resulted in a persistent SMC inflammatory phenotype that was dependent on SMC PTEN and SDF-1α expression. CONCLUSION: Resident SMC play a multifaceted role in neointima formation by contributing the majority of neointimal cells, regulating recruitment of inflammatory cells, and contributing to adventitial remodeling. The SMC PTEN-SDF-1α axis is a critical regulator of these events.

EGFR-targeted diphtheria toxin stimulates TRAIL killing of glioblastoma cells by depleting anti-apoptotic proteins.

Current treatments for Glioblastoma multiforme (GBM) involve surgery, radiotherapy, and cytotoxic chemotherapy; however, these treatments are not effective and there is an urgent need for better treatments. We investigated GBM cell killing by a novel drug combination involving DT-EGF, an Epidermal Growth Factor Receptor-targeted bacterial toxin, and Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) or antibodies that activate the TRAIL receptors DR4 and DR5. DT-EGF kills GBM cells by a non apoptotic mechanism whereas TRAIL kills by inducing apoptosis. GBM cells treated with DT-EGF and TRAIL were killed in a synergistic fashion in vitro and the combination was more effective than either treatment alone in vivo. Tumor cell death with the combination occurred by caspase activation and apoptosis due to DT-EGF positively regulating TRAIL killing by depleting FLIP, a selective inhibitor of TRAIL receptor-induced apoptosis. These data provide a mechanism-based rationale for combining targeted toxins and TRAIL receptor agonists to treat GBM.

Utilizing Optimized Tools to Investigate PTM Crosstalk: Identifying Potential PTM Crosstalk of Acetylated Mitochondrial Proteins.

Post-translational modification (PTM) crosstalk is recognized as a major cell-regulatory mechanism, and studies of several proteins have validated the premise that PTMs work in concert. Previous work by our group investigated the potential PTM crosstalk on proteins in the EGFR-Ras-c-Fos axis by utilizing a comprehensive set of PTM reagents termed Signal-Seeker toolkits. In this study, these tools were used to investigate the potential PTM crosstalk that occurs in acetylated mitochondrial proteins in response to a mitochondrial stress-inducing agent hydrogen peroxide (H2O2). Mitochondrial protein acetylation has been shown to participate in PTM crosstalk as exemplified by the regulation of the pyruvate dehydrogenase complex via kinase, phosphatase, acetyltransferase, and deacetylase activities. Changes in the acetylated state of mitochondrial proteins were investigated, in response to H2O2, using a novel anti acetyl lysine (Ac-K) antibody. Signal-Seeker PTM detection tools were used to validate the acetylation state of ten mitochondrial targets, as well as their endogenous acetylation state in response to H2O2. Importantly, the endogenous acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation state of four target mitochondrial proteins were also investigated with the toolkit. Each of the four proteins had unique PTM profiles, but diverging acetylation and ubiquitin or SUMO 2/3 signals appeared to be a common theme. This proof-of-concept study identifies the Signal-Seeker toolkits as a useful tool to investigate potential PTM crosstalk.

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins.

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein's structure and function, which may be essential for a given protein's role both physiologically and pathologically. Enrichment of PTMs is often necessary when investigating the PTM status of a target protein, because PTMs are often transient and relatively low in abundance. Many pitfalls are encountered when enriching for a PTM of a target protein, such as buffer incompatibility, the target protein antibody is not IP-compatible, loss of PTM signal, and others. The degree of difficulty is magnified when investigating multiple PTMs like acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation for a given target protein. Studying a combination of these PTMs may be necessary, as crosstalk between PTMs is prevalent and critical for protein regulation. Often, these PTMs are studied in different lysis buffers and with unique inhibitor compositions. To simplify the process, a unique denaturing lysis system was developed that effectively isolates and preserves these four PTMs; thus, enabling investigation of potential crosstalk in a single lysis system. A unique filter system was engineered to remove contaminating genomic DNA from the lysate, which is a problematic by-product of denaturing buffers. Robust affinity matrices targeting each of the four PTMs were developed in concert with the buffer system to maximize the enrichment and detection of the endogenous states of these four PTMs. This comprehensive PTM detection toolset streamlines the process of obtaining critical information about whether a protein is modified by one or more of these PTMs.

A simple toolset to identify endogenous post-translational modifications for a target protein: a snapshot of the EGFR signaling pathway.

Identification of a novel post-translational modification (PTM) for a target protein, defining its physiologic role, and studying its potential crosstalk with other PTMs is a challenging process. A set of highly sensitive tools termed Signal-Seeker kits was developed, which enables rapid and simple detection of post-translational modifications on any target protein.  The methodology for these tools utilizes affinity purification of modified proteins from a cell or tissue lysate and immunoblot analysis. These tools utilize a single lysis system that is effective at identifying endogenous, dynamic PTM changes, as well as the potential crosstalk between PTMs. As a proof-of-concept experiment, the acetylation, tyrosine phosphorylation, SUMOylation 2/3, and ubiquitination profiles of the EGFR - Ras - c-Fos axis were examined in response to EGF stimulation. All 10 previously identified PTMs of this signaling axis were confirmed using these tools, and it also identified acetylation as a novel modification of c-Fos. This axis in the EGF/EGFR signaling pathway was chosen because it is a well-established signaling pathway with proteins localized in the membrane, cytoplasmic, and nuclear compartments that ranged in abundance from 4.18x108 (EGFR) to 1.35x104 (c-Fos) molecules per A431 cell. These tools enabled the identification of low abundance PTMs, such as c-Fos Ac, at 17 molecules per cell. These studies highlight how pervasive PTMs are, and how stimulants like EGF induce multiple PTM changes on downstream signaling axis. Identification of endogenous changes and potential crosstalk between multiple PTMs for a target protein or signaling axis will provide regulatory mechanistic insight to investigators.

Identifying Regulatory Posttranslational Modifications of PD-L1: A Focus on Monoubiquitinaton.

A set of high-affinity, high-specificity posttranslational modification (PTM) enrichment tools was developed to generate an unbiased snapshot of four key PTM profiles (tyrosine phosphorylation, acetylation, ubiquitination, and SUMOylation 2/3) for the clinically important protein programmed cell death ligand 1 (PD-L1). The results showed that epidermal growth factor (EGF) treatment induced tyrosine phosphorylation, acetylation, and ubiquitination of PD-L1. Further characterization of EGF-induced PD-L1 ubiquitination revealed a significant increase in mono- and multiubiquitination of PD-L1 that occurred on glycosylated PD-L1. EGF induced mono- and multiubiquitination of PD-L1 preceded EGF-induced increases in PD-L1 protein levels. Chemical inhibitors of the EGFR pathway, gefitnib and SCH772984, suppressed PD-L1 mono- and multiubiquitination, and inhibition of the ubiquitin E1 activating enzyme, with the chemical inhibitor PYR41, was sufficient to block EGF-stimulated increases in PD-L1 protein levels. This study highlights the significance of identifying novel PTMs for PD-L1 and reveals potentially critical regulatory mechanisms that may be valuable therapeutic targets. In a broader context, this report validates an approach whereby one can gain insight into novel mechanisms of action by a simple and unbiased analysis of a PTM profile of potentially any endogenous protein of interest.

Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation.

Vascular disease progression is associated with marked changes in vascular smooth muscle cell (SMC) phenotype and function. SMC contractile gene expression and, thus differentiation, is under direct transcriptional control by the transcription factor, serum response factor (SRF); however, the mechanisms dynamically regulating SMC phenotype are not fully defined. Here we report that the lipid and protein phosphatase, PTEN, has a novel role in the nucleus by functioning as an indispensible regulator with SRF to maintain the differentiated SM phenotype. PTEN interacts with the N-terminal domain of SRF and PTEN-SRF interaction promotes SRF binding to essential promoter elements in SM-specific genes. Factors inducing phenotypic switching promote loss of nuclear PTEN through nucleo-cytoplasmic translocation resulting in reduced myogenically active SRF, but enhanced SRF activity on target genes involved in proliferation. Overall decreased expression of PTEN was observed in intimal SMCs of human atherosclerotic lesions underlying the potential clinical importance of these findings.

Selective inactivation of PTEN in smooth muscle cells synergizes with hypoxia to induce severe pulmonary hypertension.

BACKGROUND: Pulmonary vascular remodeling in pulmonary hypertension (PH) is characterized by increased vascular smooth muscle cell (SMC) and adventitial fibroblast proliferation, small vessel occlusion, and inflammatory cell accumulation. The underlying molecular mechanisms driving progression remain poorly defined. We have focused on loss of the phosphatase PTEN in SMCs as a major driver of pathological vascular remodeling. Our goal was to define the role of PTEN in human PH and in hypoxia-induced PH using a mouse model with inducible deletion of PTEN in SMCs. METHODS AND RESULTS: Staining of human biopsies demonstrated enhanced inactive PTEN selectively in the media from hypertensive patients compared to controls. Mice with induced deletion of PTEN in SMCs were exposed to normoxia or hypoxia for up to 4 weeks. Under normoxia, SMC PTEN depletion was sufficient to induce features of PH similar to those observed in wild-type mice exposed to chronic hypoxia. Under hypoxia, PTEN depletion promoted an irreversible progression of PH characterized by increased pressure, extensive pulmonary vascular remodeling, formation of complex vascular lesions, and increased macrophage accumulation associated with synergistic increases in proinflammatory cytokines and proliferation of both SMCs and nonSMCs. CONCLUSIONS: Chronic inactivation of PTEN selectively in SMC represents a critical mediator of PH progression, leading to cell autonomous events and increased production of factors correlated to proliferation and recruitment of adventitial and inflammatory cells, resulting in irreversible progression of the disease.

Inactivation of the tumour suppressor, PTEN, in smooth muscle promotes a pro-inflammatory phenotype and enhances neointima formation.

AIMS: Phosphatase and tensin homolog (PTEN) is implicated as a negative regulator of vascular smooth muscle cell (SMC) proliferation and injury-induced vascular remodelling. We tested if selective depletion of PTEN only in SMC is sufficient to promote SMC phenotypic modulation, cytokine production, and enhanced neointima formation. METHODS AND RESULTS: Smooth muscle marker expression and induction of pro-inflammatory cytokines were compared in cultured SMC expressing control or PTEN-specific shRNA. Compared with controls, PTEN-deficient SMC exhibited increased phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signalling and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) activity, reduced expression of SM markers (SM-alpha-actin and calponin), and increased production of stromal cell-derived factor-1alpha (SDF-1alpha), monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), and chemokine (C-X-C motif) ligand 1 (KC/CXCL1) under basal conditions. PI3K/Akt or mTOR inhibition reversed repression of SM marker expression, whereas PI3K/Akt or NF-kappaB inhibition blocked cytokine induction mediated by PTEN depletion. Carotid ligation in mice with genetic reduction of PTEN specifically in SMC (SMC-specific PTEN heterozygotes) resulted in enhanced neointima formation, increased SMC hyperplasia, reduced SM-alpha-actin and calponin expression, and increased NF-kappaB and cytokine expression compared with wild-types. Lesion formation in SMC-specific heterozygotes was similar to lesion formation in global PTEN heterozygotes, indicating that inactivation of PTEN exclusively in SMC is sufficient to induce considerable increases in neointima formation. CONCLUSION: PTEN activation specifically in SMC is a common upstream regulator of multiple downstream events involved in pathological vascular remodelling, including proliferation, de-differentiation, and production of multiple cytokines.

Acute myeloid leukemia-targeted toxin activates both apoptotic and necroptotic death mechanisms.

BACKGROUND: Acute myelogenous leukemia (AML) is the second most common leukemia with approximately 13,410 new cases and 8,990 deaths annually in the United States. A novel fusion toxin treatment, diphtheria toxin GM-CSF (DT-GMCSF) has been shown to selectively eliminate leukemic repopulating cells that are critical for the formation of AML. We previously showed that DT-GMCSF treatment of U937 cells, an AML cell line, causes activation of caspases and the induction of apoptosis. METHODS AND FINDINGS: In this study we further investigate the mechanisms of cell death induced by DT-GMCSF and show that, in addition to the activation of caspase-dependent apoptosis, DT-GMCSF also kills AML cells by simultaneously activating caspase-independent necroptosis. These mechanisms depend on the ability of the targeted toxin to inhibit protein synthesis, and are not affected by the receptor that is targeted or the mechanism through which protein synthesis is blocked. CONCLUSIONS: We conclude that fusion toxin proteins may be effective for treating AML cells whether or not they are defective in apoptosis.