The metabolic ER stress sensor IRE1α suppresses alternative activation of macrophages and impairs energy expenditure in obesity.

Obesity is associated with metabolic inflammation and endoplasmic reticulum (ER) stress, both of which promote metabolic disease progression. Adipose tissue macrophages (ATMs) are key players orchestrating metabolic inflammation, and ER stress enhances macrophage activation. However, whether ER stress pathways underlie ATM regulation of energy homeostasis remains unclear. Here, we identified inositol-requiring enzyme 1α (IRE1α) as a critical switch governing M1-M2 macrophage polarization and energy balance. Myeloid-specific IRE1α abrogation in Ern1f/f; Lyz2-Cre mice largely reversed high-fat diet (HFD)-induced M1-M2 imbalance in white adipose tissue (WAT) and blocked HFD-induced obesity, insulin resistance, hyperlipidemia and hepatic steatosis. Brown adipose tissue (BAT) activity, WAT browning and energy expenditure were significantly higher in Ern1f/f; Lyz2-Cre mice. Furthermore, IRE1α ablation augmented M2 polarization of macrophages in a cell-autonomous manner. Thus, IRE1α senses protein unfolding and metabolic and immunological states, and consequently guides ATM polarization. The macrophage IRE1α pathway drives obesity and metabolic syndrome through impairing BAT activity and WAT browning.

IRE1α inhibits osteogenic differentiation of mouse embryonic fibroblasts by limiting Shh signaling.

OBJECTIVES: This study aimed to investigate the effect of endoplasmic reticulum (ER) stress sensor inositol-requiring enzyme 1α (IRE1α) on the sonic hedgehog N-terminus (N-Shh)-enhanced-osteogenic differentiation process in mouse embryonic fibroblasts (MEFs). MATERIALS AND METHODS: Osteogenesis of MEFs was observed by alkaline phosphatase (ALP) staining, alizarin red staining, and Von Kossa staining assays. Activation of unfolded protein response and Shh signaling were examined using real-time quantitative PCR and western blot assays. IRE1α-deficient MEFs were used to explore the effect of IRE1α on N-Shh-driven osteogenesis. RESULTS: N-Shh increased ALP activity, matrix mineralization, and the expression of Alp and Col-I in MEFs under osteogenic conditions; notably, this was reversed when combined with the ER stress activator Tm treatment. Interestingly, the administration of N-Shh decreased the expression of IRE1α. Abrogation of IRE1α increased the expression of Shh pathway factors in osteogenesis-induced MEFs, contributing to the osteogenic effect of N-Shh. Moreover, IRE1α-deficient MEFs exhibited elevated levels of osteogenic markers. CONCLUSIONS: Our findings suggest that the IRE1α-mediated unfolded protein response may alleviate the ossification of MEFs by attenuating Shh signaling. Our research has identified a strategy to inhibit excessive ossification, which may have clinical significance in preventing temporomandibular joint bony ankylosis.

Phosphorylation at Ser724 of the ER stress sensor IRE1α governs its activation state and limits ER stress-induced hepatosteatosis.

Inositol-requiring enzyme 1 (IRE1) is an evolutionarily conserved sensor of endoplasmic reticulum (ER) stress and mediates a key branch of the unfolded protein response in eukaryotic cells. It is an ER-resident transmembrane protein that possesses Ser/Thr protein kinase and endoribonuclease (RNase) activities in its cytoplasmic region. IRE1 is activated through dimerization/oligomerization and autophosphorylation at multiple sites, acting through its RNase activity to restore the functional capacity of the ER. However, it remains poorly defined in vivo how the autophosphorylation events of endogenous IRE1 govern its dynamic activation and functional output. Here, we generated a mouse model harboring a S724A knock-in mutation (Ern1S724A/S724A) and investigated the importance of phosphorylation at Ser724 within the kinase activation loop of murine IRE1α. We found that in mouse embryonic fibroblast cells and in primary hepatocytes, S724A mutation resulted in markedly reduced IRE1α autophosphorylation in parallel with blunted activation of its RNase activity to catalyze X-box binding protein 1 (Xbp1) mRNA splicing. Furthermore, ablation of IRE1α phosphorylation at Ser724 exacerbated ER stress-induced hepatic steatosis in tunicamycin-treated Ern1S724A/S724A mice. This was accompanied by significantly decreased hepatic production of spliced XBP1 protein but increased CCAAT-enhancer-binding protein homologous protein (CHOP) level, along with suppressed expression of key metabolic regulators of fatty acid ß-oxidation and lipid secretion. These results demonstrate a critical role of phosphorylation at Ser724 of IRE1α in dynamically controlling its kinase activity, and thus its autophosphorylation state, which is coupled to activation of its RNase activity in counteracting hepatic steatosis under ER stress conditions.

The ER stress sensor inositol-requiring enzyme 1α in Kupffer cells promotes hepatic ischemia-reperfusion injury.

Hepatic ischemia/reperfusion (I/R) injury is an inflammation-mediated process arising from ischemia/reperfusion-elicited stress in multiple cell types, causing liver damage during surgical procedures and often resulting in liver failure. Endoplasmic reticulum (ER) stress triggers the activation of the unfolded protein response (UPR) and is implicated in tissue injuries, including hepatic I/R injury. However, the cellular mechanism that links the UPR signaling to local inflammatory responses during hepatic I/R injury remains largely obscure. Here, we report that IRE1α, a critical ER-resident transmembrane signal transducer of the UPR, plays an important role in promoting Kupffer-cell-mediated liver inflammation and hepatic I/R injury. Utilizing a mouse model in which IRE1α is specifically ablated in myeloid cells, we found that abrogation of IRE1α markedly attenuated necrosis and cell death in the liver, accompanied by reduced neutrophil infiltration and liver inflammation following hepatic I/R injury. Mechanistic investigations in mice as well as in primary Kupffer cells revealed that loss of IRE1α in Kupffer cells not only blunted the activation of the NLRP3 inflammasome and IL-1ß production, but also suppressed the expression of the inducible nitric oxide synthase (iNos) and proinflammatory cytokines. Moreover, pharmacological inhibition of IRE1α's RNase activity was able to attenuate inflammasome activation and iNos expression in Kupffer cells, leading to alleviation of hepatic I/R injury. Collectively, these results demonstrate that Kupffer cell IRE1α mediates local inflammatory damage during hepatic I/R injury. Our findings suggest that IRE1α RNase activity may serve as a promising target for therapeutic treatment of ischemia/reperfusion-associated liver inflammation and dysfunction.

Hypoxic ER stress suppresses ß-catenin expression and promotes cooperation between the transcription factors XBP1 and HIF1α for cell survival.

Hypoxia occurs in many human solid tumors and activates multiple cellular adaptive-response pathways, including the unfolded protein response (UPR) in the endoplasmic reticulum (ER). Wnt/ß-catenin signaling plays a critical role in tumorigenesis, and ß-catenin has been shown to enhance hypoxia-inducible factor 1α (HIF1α)-activated gene expression, thereby supporting cell survival during hypoxia. However, the molecular interplay between hypoxic ER stress, Wnt/ß-catenin signaling, and HIF1α-mediated gene regulation during hypoxia remains incompletely understood. Here, we report that hypoxic ER stress reduces ß-catenin stability, which, in turn, enhances the activity of spliced X-box-binding protein 1 (XBP1s), a transcription factor and signal transducer of the UPR, in HIF1α-mediated hypoxic responses. We observed that in the RKO colon cancer cell line, which possesses a Wnt-stimulated ß-catenin signaling cascade, increased ER stress during hypoxia is accompanied by a reduction in low-density lipoprotein receptor-related protein 6 (LRP6), and this reduction in LRP6 decreased ß-catenin accumulation and impaired Wnt/ß-catenin signaling. Of note, ß-catenin interacted with both XBP1s and HIF1α, suppressing XBP1s-mediated augmentation of HIF1α target gene expression. Furthermore, Wnt stimulation or ß-catenin overexpression blunted XBP1s-mediated cell survival under hypoxia. Together, these results reveal an unanticipated role for the Wnt/ß-catenin pathway in hindering hypoxic UPR-mediated responses that increase cell survival. Our findings suggest that the molecular cross-talks between hypoxic ER stress, LRP6/ß-catenin signaling, and the HIF1α pathway may represent an unappreciated mechanism that enables some tumor subtypes to survive and grow in hypoxic conditions.

Dual role for inositol-requiring enzyme 1α in promoting the development of hepatocellular carcinoma during diet-induced obesity in mice.

Obesity is associated with both endoplasmic reticulum (ER) stress and chronic metabolic inflammation. ER stress activates the unfolded protein response (UPR) and has been implicated in a variety of cancers, including hepatocellular carcinoma (HCC). It is unclear whether individual UPR pathways are mechanistically linked to HCC development, however. Here we report a dual role for inositol-requiring enzyme 1α (IRE1α), the ER-localized UPR signal transducer, in obesity-promoted HCC development. We found that genetic ablation of IRE1α in hepatocytes not only markedly reduced the occurrence of diethylnitrosamine (DEN)-induced HCC in liver-specific IRE1α knockout (LKO) mice when fed a normal chow (NC) diet, but also protected against the acceleration of HCC progression during high-fat diet (HFD) feeding. Irrespective of their adiposity states, LKO mice showed decreased hepatocyte proliferation and signal transducer and activator of transcription 3 (STAT3) activation, even in the face of increased hepatic apoptosis. Furthermore, IRE1α abrogation blunted obesity-associated activation of hepatic inhibitor of nuclear factor kappa B kinase subunit beta (IKKß)-nuclear factor kappa B (NF-κB) pathway, leading to reduced production of the tumor-promoting inflammatory cytokines tumor necrosis factor (TNF) and interleukin 6 (IL-6). Importantly, higher IRE1α expression along with elevated STAT3 phosphorylation was also observed in the tumor tissues from human HCC patients, correlating with their poorer survival rate. CONCLUSION: IRE1α acts in a feed-forward loop during obesity-induced metabolic inflammation to promote HCC development through STAT3-mediated hepatocyte proliferation. (Hepatology 2018).

Brain-expressed X-linked 2 Is Pivotal for Hyperactive Mechanistic Target of Rapamycin (mTOR)-mediated Tumorigenesis.

Frequent alteration of upstream proto-oncogenes and tumor suppressor genes activates mechanistic target of rapamycin (mTOR) and causes cancer. However, the downstream effectors of mTOR remain largely elusive. Here we report that brain-expressed X-linked 2 (BEX2) is a novel downstream effector of mTOR. Elevated BEX2 in Tsc2(-/-) mouse embryonic fibroblasts, Pten(-/-) mouse embryonic fibroblasts, Tsc2-deficient rat uterine leiomyoma cells, and brains of neuronal specific Tsc1 knock-out mice were abolished by mTOR inhibitor rapamycin. Furthermore, BEX2 was also increased in the liver of a hepatic specific Pten knock-out mouse and the kidneys of Tsc2 heterozygous deletion mice, and a patient with tuberous sclerosis complex (TSC). mTOR up-regulation of BEX2 was mediated in parallel by both STAT3 and NF-κB. BEX2 was involved in mTOR up-regulation of VEGF production and angiogenesis. Depletion of BEX2 blunted the tumorigenesis of cells with activated mTOR. Therefore, enhanced STAT3/NF-κB-BEX2-VEGF signaling pathway contributes to hyperactive mTOR-induced tumorigenesis. BEX2 may be targeted for the treatment of the cancers with aberrantly activated mTOR signaling pathway.

Reduction-responsive cholesterol-based block copolymer vesicles for drug delivery.

We developed a new robust reduction-responsive polymersome based on the amphiphilic block copolymer PEG-SS-PAChol. The stability and robustness were achieved by the smectic physical cross-linking of cholesterol-containing liquid crystal polymer PAChol in the hydrophobic layer. The reduction-sensitivity was introduced by the disulfide bridge (-S-S-) that links the hydrophilic PEG block and the hydrophobic PAChol block. We used a versatile synthetic strategy based on atom transfer radical polymerization (ATRP) to synthesize the reduction-responsive amphiphilic block copolymers. The reductive cleavage of the disulfide bridge in the block copolymers was first evidenced in organic solution. The partial destruction of PEG-SS-PAChol polymersomes in the presence of a reducing agent was then demonstrated by cryo-electron microscopy. Finally, the calcein release from PEG-SS-PAChol polymersomes triggered by glutathione (GSH) was observed both in PBS suspension and in vitro inside the macrophage cells. High GSH concentrations (≥35 mM in PBS or artificially enhanced in macrophage cells by GSH-OEt pretreatment) and long incubation time (in the order of hours) were, however, necessary to get significant calcein release. These polymersomes could be used as drug carriers with very long circulation profiles and slow release kinetics.

Mammalian IRE1α dynamically and functionally coalesces with stress granules.

Upon endoplasmic reticulum (ER) stress, activation of the ER-resident transmembrane protein kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1) initiates a key branch of the unfolded protein response (UPR) through unconventional splicing generation of the transcription factor X-box-binding protein 1 (XBP1s). Activated IRE1 can form large clusters/foci, whose exact dynamic architectures and functional properties remain largely elusive. Here we report that, in mammalian cells, formation of IRE1α clusters is an ER membrane-bound phase separation event that is coupled to the assembly of stress granules (SGs). In response to different stressors, IRE1α clusters are dynamically tethered to SGs at the ER. The cytosolic linker portion of IRE1α possesses intrinsically disordered regions and is essential for its condensation with SGs. Furthermore, disruption of SG assembly abolishes IRE1α clustering and compromises XBP1 mRNA splicing, and such IRE1α-SG coalescence engenders enrichment of the biochemical components of the pro-survival IRE1α-XBP1 pathway during ER stress. Our findings unravel a phase transition mechanism for the spatiotemporal assembly of IRE1α-SG condensates to establish a more efficient IRE1α machinery, thus enabling higher stress-handling capacity.

Polymersomes with PEG corona: structural changes and controlled release induced by temperature variation.

Thermoresponsive behavior of different kinds of polymersomes was studied using small angle neutron scattering (SANS), transmission electron microscopy (TEM), and proton nuclear magnetic resonance ((1)H NMR). The polymersomes were made of block copolymers containing a 2000 Da polyethylene glycol (PEG) as a hydrophilic block and either a liquidlike polymer (e.g., PBA: polybutylacrylate), a solidlike polymer (PS: polystyrene), or a liquid crystalline (LC) polymer as a hydrophobic block. Structural changes in polymersomes are driven in all cases by the critical dehydration temperature of PEG corona, which is closely related to the chemical structure and chain mobility of the hydrophobic block. No structural changes occur upon heating from 25 to 75 °C in the liquidlike polymersomes where the critical dehydration temperature of PEG should be higher than 75 °C. In contrast, glassy PEG-b-PS polymersomes and LC polymersomes show structural changes around 55 °C, which corresponds to the critical dehydration temperature of PEG in those block copolymers. Furthermore, the structural changes depend on the properties of the hydrophobic layer. Glassy PEG-b-PS polymersomes aggregate together above 55 °C, but the bilayer membrane is robust enough to remain intact. This aggregation is reversible, and rather separate polymersomes are recovered upon cooling. However, LC polymersomes display drastic and irreversible structural changes when heated above ∼55 °C. These changes are dependent on the LC structures of the hydrophobic layer. Nematic LC polymersomes turn into thick-walled capsules, whereas smectic LC polymersomes collapse into dense aggregates. As these drastic and irreversible changes decrease or remove the inner compartment volume of the vesicle, LC polymersomes can be used for thermal-responsive controlled release, as shown by a study of calcein release. Finally, toxicity studies proved that LC polymersomes were noncytotoxic and had no effect on cell morphology.

Adipocyte IRE1α promotes PGC1α mRNA decay and restrains adaptive thermogenesis.

Adipose tissue undergoes thermogenic remodeling in response to thermal stress and metabolic cues, playing a crucial role in regulating energy expenditure and metabolic homeostasis. Endoplasmic reticulum (ER) stress is associated with adipose dysfunction in obesity and metabolic disease. It remains unclear, however, if ER stress-signaling in adipocytes mechanistically mediates dysregulation of thermogenic fat. Here we show that inositol-requiring enzyme 1α (IRE1α), a key ER stress sensor and signal transducer, acts in both white and beige adipocytes to impede beige fat activation. Ablation of adipocyte IRE1α promotes browning/beiging of subcutaneous white adipose tissue following cold exposure or ß3-adrenergic stimulation. Loss of IRE1α alleviates diet-induced obesity and augments the anti-obesity effect of pharmacologic ß3-adrenergic stimulation. Notably, IRE1α suppresses stimulated lipolysis and degrades Ppargc1a messenger RNA through its RNase activity to downregulate the thermogenic gene program. Hence, blocking IRE1α bears therapeutic potential in unlocking adipocytes' thermogenic capacity to combat obesity and metabolic disorders.

IRE1α regulates skeletal muscle regeneration through Myostatin mRNA decay.

Skeletal muscle can undergo a regenerative process from injury or disease to preserve muscle mass and function, which is critically influenced by cellular stress responses. Inositol-requiring enzyme 1 (IRE1) is an ancient endoplasmic reticulum (ER) stress sensor and mediates a key branch of the unfolded protein response (UPR). In mammals, IRE1α is implicated in the homeostatic control of stress responses during tissue injury and regeneration. Here, we show that IRE1α serves as a myogenic regulator in skeletal muscle regeneration in response to injury and muscular dystrophy. We found in mice that IRE1α was activated during injury-induced muscle regeneration, and muscle-specific IRE1α ablation resulted in impaired regeneration upon cardiotoxin-induced injury. Gain- and loss-of-function studies in myocytes demonstrated that IRE1αacts to sustain both differentiation in myoblasts and hypertrophy in myotubes through regulated IRE1-dependent decay (RIDD) of mRNA encoding Myostatin, a key negative regulator of muscle repair and growth. Furthermore, in the mouse model of Duchenne muscular dystrophy (DMD), loss of muscle IRE1α resulted in augmented Myostatin signaling and exacerbated the dystrophic phenotypes. Thus, these results reveal a pivotal role for the RIDD output of IRE1α in muscle regeneration, offering new insight into potential therapeutic strategies for muscle loss diseases.

Cyclin K and cyclin D1b are oncogenic in myeloma cells.

BACKGROUND: Aberrant expression of cyclin D1 is a common feature in multiple myeloma (MM) and always associated with mantle cell lymphoma (MCL). CCND1 gene is alternatively spliced to produce two cyclin D1 mRNA isoforms which are translated in two proteins: cyclin D1a and cyclin D1b. Both isoforms are present in MM cell lines and primary cells but their relative role in the tumorigenic process is still elusive. RESULTS: To test the tumorigenic potential of cyclin D1b in vivo, we generated cell clones derived from the non-CCND1 expressing MM LP-1 cell line, synthesizing either cyclin D1b or cyclin K, a structural homolog and viral oncogenic form of cyclin D1a. Immunocompromised mice injected s.c. with LP-1K or LP-1D1b cells develop tumors at the site of injection. Genome-wide analysis of LP-1-derived cells indicated that several cellular processes were altered by cyclin D1b and/or cyclin K expression such as cell metabolism, signal transduction, regulation of transcription and translation. Importantly, cyclin K and cyclin D1b have no major action on cell cycle or apoptosis regulatory genes. Moreover, they impact differently cell functions. Cyclin K-expressing cells have lost their migration properties and display enhanced clonogenic capacities. Cyclin D1b promotes tumorigenesis through the stimulation of angiogenesis. CONCLUSIONS: Our study indicates that cyclin D1b participates into MM pathogenesis via previously unrevealed actions.

A novel iodomethylene-dimethyl-dihydropyranone induces G2/M arrest and apoptosis in human cancer cells.

BACKGROUND: The putative pharmacophore of a naturally cytotoxic limonoid haperforin B1, E-5-iodomethylene-6,6-dimethyl-5,6-dihydropyran-2-one (IDDP) was synthesized and its biological activity was investigated. MATERIALS AND METHODS: The cytotoxicity of IDDP was assessed using human breast, lung, colorectal and epidermal carcinomas, chronic myeloid leukemia and glioblastoma cell lines. Cell cycle analysis was performed by flow cytometry. The induction of apoptosis was studied by a caspase assay and by annexin V-propidium iodide double staining. The organization of actin and tubulin microfilaments was analysed by immunocytochemical labeling. RESULTS: IDDP was shown to inhibit the growth of a panel of human cancer cell lines independently of their p53 status with IC(50) ranging from 0.07 to 0.50 microM. All the treated cells were arrested in the G(2)/M phase in a time-dependent manner before cell death occurred through an apoptotic pathway. Immunocytochemical studies revealed that the normal organization of microfilaments and microtubules was disrupted in IDDP exposed cells. CONCLUSION: IDDP can be considered as a promising anticancer agent.

New C5-alkylated indolobenzazepinones acting as inhibitors of tubulin polymerization: cytotoxic and antitumor activities.

A series of 5-alkylindolobenzazepin-7-ones was synthesized by Suzuki coupling between 3-iodoindole-2-carboxylates and the appropriate alpha-alkylbenzylamino o-boronic acids followed by cyclization to the lactam. Derivatives having a linear alkyl chain at C5 were found to be highly cytotoxic to KB cells with IC50 values in the 30-80 nM range. These compounds also inhibited the polymerization of tubulin with IC50's of 1-2 microM. Compound 4f (( S)-5-ethyl) showed comparable antiproliferative activities (IC50's of 30-70 nM) in a variety of cancer cell lines, cell growth being arrested at the G2/M phase. Compound 4f induced apoptosis in a dose-dependent manner in three different cancer cell lines and was shown to affect cell morphology in a manner consistent with its inhibitory action on tubulin polymerization. Using the experimental model of glioma grafted on the chick chorio-allantoic membrane, local treatment with compound 4f markedly reduced tumor progression.

Synthesis and antitumor activity of benzils related to combretastatin A-4.

A series of benzil derivatives related to combretastatin A-4 (CA-4) have been synthesized by oxidation of diarylalkynes promoted by PdI(2) in DMSO. Using this new protocol, 14 benzils were prepared in good to excellent yields and their biological activity has been delineated. Several benzils exhibited excellent antiproliferative activity: for example, 4j and 4k bearing the greatest resemblance to CA-4 and AVE-8062, respectively, were found to inhibit cell growth at the nanomolar level (20-50nM) on four human tumor cell lines. Flow cytometric analysis indicates that these compounds act as antimitotics and arrest the cell cycle in G(2)/M phase. A cell-based assay indicated that compounds 4j and 4k displayed a similar inhibition of tubulin assembly with an IC(50) value similar to CA-4. These results clearly demonstrated that the Z-double bond of CA-4 can be replaced by a 1,2-diketone unit without significant loss of cytotoxicity and inhibition of tubulin assembly potency.

Overexpression of the angiogenic tetrapeptide AcSDKP in human malignant tumors.

BACKGROUND: The natural tetrapeptide acetyl-Ser-Asp-Lys-Pro (AcSDKP), generated from thymosin beta4 following its cleavage by prolyl oligopeptidase (POP), is a physiological stimulator of angiogenesis. Because of the critical role of neovascularisation in tumor development, the expression of AcSDKP and the activity of POP were examined in different human solid malignancies. MATERIALS AND METHODS: The expression of AcSDKP and the activity of POP were evaluated in human blood samples and tissue specimens of thyroid goiter and thyroid papillary carcinoma as well as in commercial cancer tissue microarray. RESULTS: A significantly increased concentration of AcSDKP in intratumoral blood and enhanced tissular activity of POP were detected in cancer patients. The expression of AcSDKP in human breast, colon, head and neck, kidney, lung, skin, ovary and prostate cancer tissues was shown to be greater than that in normal tissues. CONCLUSION: AcSDKP and POP contribute to the malignant phenotype and these molecules are potentiel markers of cancer.

Inhibition of neddylation by MLN4924 improves neointimal hyperplasia and promotes apoptosis of vascular smooth muscle cells through p53 and p62.

Targeting apoptosis of vascular smooth muscle cells (VSMCs) represents an attractive approach to diminish the occurrence of restenosis. Neddylation is a highly conserved post-translational modification process and inhibition of neddylation has been shown to regulate apoptosis of other cells. However, the impacts of neddylation inhibition on VSMCs and neointimal hyperplasia have not been studied. In our present study, we have shown that MLN4924, a selective inhibitor of NEDD8-activating enzyme (NAE), markedly inhibited neointimal hyperplasia and accumulation of VSMCs, whereas increased apoptosis in the vascular wall. In vitro studies revealed that MLN4924 induced G2/M arrest and apoptosis of human VSMCs. Knockdown of NAE1 had similar effects. MLN4924 upregulated p53 and p62 in human VSMCs. Knockdown of either p53 or p62 mitigated the impacts of MLN4924 on G2/M arrest and apoptosis. Moreover, p53 knockdown abolished MLN4924-induced upregulation of p62. Finally, smooth muscle p53 knockout mice were generated and subjected to femoral artery injury and MLN4924 treatment. Deficiency of p53 in smooth muscle blocked the effects of MLN4924 on neointimal hyperplasia and apoptosis. Together, our results revealed that neddylation inhibition induces apoptosis through p53 and p62 in VSMCs and improves neointimal hyperplasia mainly by promoting apoptosis through smooth muscle p53 in mice. These pre-clinical data provide strong translational implications for targeting restenosis by perturbation of neddylation using MLN4924.

Tetrapeptide AcSDKP induces postischemic neovascularization through monocyte chemoattractant protein-1 signaling.

BACKGROUND: We investigated the putative proangiogenic activity and molecular pathway(s) of the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) in a model of surgically induced hindlimb ischemia. METHODS AND RESULTS: Hindlimb ischemia was induced by femoral artery ligature and an osmotic minipump was implanted subcutaneously to deliver low (0.12 mg/kg per day) or high (1.2 mg/kg per day) doses of AcSDKP, for 7 or 21 days. Angiography scores, arteriole density, capillary number, and foot perfusion were increased at day 21 in the high-dose AcSDKP-treated mice (by 1.9-, 1.8-, 1.3-, and 1.6-fold, respectively) compared with control animals (P<0.05, P<0.01, P<0.01, respectively). AcSDKP treatment for 24 hours upregulated the monocyte chemoattractant protein-1 (MCP-1) mRNA and protein levels by 1.5-fold in cultured endothelial cells (P<0.01). In the ischemic hindlimb model, administration of AcSDKP also enhanced MCP-1 mRNA levels by 90-fold in ischemic leg (P<0.001) and MCP-1 plasma levels by 3-fold (P<0.001 versus untreated ischemic control mice). MCP-1 levels upregulation were associated with a 2.3-fold increase in the number of Mac3-positive cells in ischemic area of AcSDKP-treated mice (P<0.001 versus untreated animals). Interestingly, AcSDKP-induced monocyte/macrophage infiltration and postischemic neovascularization was fully blunted in MCP-1-deficient animals. CONCLUSIONS: AcSDKP stimulates postischemic neovascularization through activation of a proinflammatory MCP-1-related pathway.

Evidence for an association of high levels of endogenous Acetyl-Ser-Asp-Lys-Pro, a potent mediator of angiogenesis, with acute myeloid leukemia development.

Evidence from clinical and laboratory studies suggests that angiogenesis is important in the progression of solid tumours and hematologic malignancies. We have shown that the naturally occurring tetrapeptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP) is a potent angiogenic factor normally present at nanomolar concentrations in the blood. A murine leukemia model was used to assess whether there was a correlation between levels of endogenous AcSDKP and the development of disease. Levels of AcSDKP in the plasma and bone marrow (BM) cells from mice bearing an acute myeloid leukemia (AML) were five- to ten-fold greater than those in non-leukemic mice. Furthermore, a strong correlation between the concentration of endogenous AcSDKP and the progression of AML was demonstrated. These results are consistent with the marked increase in BM vascularity observed in leukemic mice. The physiologic relevance of these findings awaits further studies and the contribution of AcSDKP to the pathogenesis of leukemia is under investigation.