Long-term correction of hemorrhagic diathesis in hemophilia A mice by an AAV-delivered hybrid FVIII composed of the human heavy chain and the rat light chain.

Conventional therapies for hemophilia A (HA) are prophylactic or on-demand intravenous FVIII infusions. However, they are expensive and inconvenient to perform. Thus, better strategies for HA treatment must be developed. In this study, a recombinant FVIII cDNA encoding a human/rat hybrid FVIII with an enhanced procoagulant potential for adeno-associated virus (AAV)-delivered gene therapy was developed. Plasmids containing human FVIII heavy chain (hHC), human light chain (hLC), and rat light chain (rLC) were transfected into cells and hydrodynamically injected into HA mice. Purified AAV viruses were intravenously injected into HA mice at two doses. Results showed that the hHC + rLC protein had a higher activity than the hHC + hLC protein at comparable expression levels. The specific activity of hHC + rLC was about 4- to 8-fold higher than that of their counterparts. Hydrodynamic injection experiments obtained consistent results. Notably, the HA mice undergoing the AAV-delivered hHC + rLC treatment exhibited a visibly higher activity than those treated with hHC + hLC, and the therapeutic effects lasted for up to 40 weeks. In conclusion, the application of the hybrid FVIII (hHC + rLC) via an AAV-delivered gene therapy substantially improved the hemorrhagic diathesis of the HA mice. These data might be of help to the development of optimized FVIII expression cassette for HA gene therapy.

Targeting the RT loop of Src SH3 in Platelets Prevents Thrombosis without Compromising Hemostasis.

Conventional antiplatelet agents indiscriminately inhibit both thrombosis and hemostasis, and the increased bleeding risk thus hampers their use at more aggressive dosages to achieve adequate effect. Blocking integrin αIIbß3 outside-in signaling by separating the ß3/Src interaction, yet to be proven in vivo, may nonetheless resolve this dilemma. Identification of a specific druggable target for this strategy remains a fundamental challenge as Src SH3 is known to be responsible for binding to not only integrin ß3 but also the proteins containing the PXXP motif. In vitro and in vivo mutational analyses show that the residues, especially E97, in the RT loop of Src SH3 are critical for interacting with ß3. DCDBS84, a small molecule resulting from structure-based virtual screening, is structurally validated to be directed toward the projected target. It specifically disrupts ß3/Src interaction without affecting canonical PXXP binding and thus inhibits the outside-in signaling-regulated platelet functions. Treatment of mice with DCDBS84 causes a profound inhibition of thrombosis, equivalent to that induced by extremely high doses of αIIbß3 antagonist, but does not compromise primary hemostasis. Specific targets are revealed for a preferential inhibition of thrombosis that may lead to new classes of potent antithrombotics without hemorrhagic side effects.

Mitochondrial activity regulates the differentiation of skin-derived mesenchymal stem cells into brown adipocytes to contribute to hypertension.

BACKGROUND: Brown adipocytes (BAs) are major components of brown adipose tissue (BAT), which is involved in blood pressure regulation. BAs are derived from multiple progenitors, including PDGFRα+ adipose-derived stem cells (ASCs). Skin-derived mesenchymal stem cells (S-MSCs) have the capacity to differentiate into adipocytes; however, their ability to differentiate into BAs remains unexplored. We aim to study the ability and regulatory mechanism of the differentiation of S-MSCs into BAs and the direct role of BAT in blood pressure regulation. METHODS: Protein expression was measured by flow cytometry or Western blotting, and gene mRNA levels were quantified by real-time quantitative PCR (RT-PCR). To induce the differentiation of S-MSCs into BAs, S-MSCs were stimulated with a brown adipogenic cocktail comprising insulin, IBMX, dexamethasone, triiodothyronine (T3), and rosiglitazone for the indicated periods. The oxygen consumption rate (OCR) was measured with an XF24 Extracellular Flux Analyzer. Mitochondrial mass was determined by flow cytometry and fluorescence staining. Hypertension was induced in WT mice by infusion of angiotensin II (Ang II), and systolic blood pressure (SBP) was measured using a tail cuff. Interscapular brown adipose tissue (iBAT)-deficient mice were generated by surgical removal of the iBAT depot, after which the animals were allowed to recover for 6 days. Aortic, iBAT, and heart tissue sections were analyzed by hematoxylin and eosin (HE) staining. RESULTS: We found that in vitro, S-MSCs isolated from the mouse dermis expressed the stem cell markers CD90/105 and PDGFRα and readily differentiated into BAs. Mitochondrial biogenesis and oxygen consumption were markedly increased during differentiation of S-MSCs into BAs. In vivo, iBAT was converted to white adipose tissue (WAT) in Ang II-induced hypertensive mice. We assessed the direct role of BAT in blood pressure (BP) regulation by using iBAT-deficient mice (generated by surgical removal of iBAT) and C57BL/6 (wild-type (WT)) mice and found that Ang II-induced BP elevation and vascular damage were markedly aggravated in iBAT-deficient mice compared with WT mice. CONCLUSIONS: This study demonstrates that PDGFRα+ S-MSCs are able to differentiate into BAs and that this differentiation is regulated by mitochondrial activity. We also show that BAT plays a direct role in ameliorating Ang II-induced hypertension. The therapeutic potential of BAT for the prevention of hypertension-induced organ remodeling thus warrants further investigation.

Integrin ß3 Deficiency Results in Hypertriglyceridemia via Disrupting LPL (Lipoprotein Lipase) Secretion.

OBJECTIVE: Integrin ß3 is implicated in numerous biological processes such as its relevance to blood triglyceride, yet whether ß3 deficiency affects this metabolic process remains unknown. Approach and Results: We showed that the Chinese patients with ß3-deficient Glanzmann thrombasthenia had a 2-fold higher serum triglyceride level together with a lower serum LPL (lipoprotein lipase) level than those with an αIIb deficiency or healthy subjects. The ß3 knockout mice recapitulated these phenotypic features. The elevated plasma triglyceride level was due to impaired LPL-mediated triglyceride clearance caused by a disrupted LPL secretion. Further analysis revealed that ß3 directly bound LPL via a juxtamembrane TIH (threonine isoleucine histidine)720-722 motif in its cytoplasmic domain and functioned as an adaptor protein by interacting with LPL and PKD (protein kinase D) to form the PKD/ß3/LPL complex that is required for ß3-mediated LPL secretion. Furthermore, the impaired triglyceride clearance in ß3 knockout mice could be corrected by adeno-associated virus serotype 9 (AAV9)-mediated delivery of wild-type but not TIH720-722-mutated ß3 genes. CONCLUSIONS: This study reveals a hypertriglyceridemia in both ß3-deficient Chinese patients and mice and provides novel insights into the molecular mechanisms of the significant roles of ß3 in LPL secretion and triglyceride metabolism, drawing attention to the metabolic consequences in patients with ß3-deficient Glanzmann thrombasthenia.

MicroRNA-31 Regulates Immunosuppression in Ang II (Angiotensin II)-induced Hypertension by Targeting Ppp6C (Protein Phosphatase 6c).

Regulatory T cells (Treg cells) play important roles in hypertension and organ damages. MicroRNA-31 (miR-31) is a critical regulator for Treg cell generation. However, the role of miR-31 in hypertension has not been elucidated. We aim to study the functionality of miR-31 and the detailed mechanism in Ang II (Angiotensin II)-induced hypertensive mouse model. We found: In vitro, miR-31 expression was higher in T helper 17 cells and lower in Treg cells than that of naïve T cells. The genetic deficiency of miR-31 promoted Treg cell differentiation, whereas no impact on T helper 17 cells differentiation. Ang II-induced hypertension resulted in increased expression of miR-31 in the aorta, splenic CD4+ T cells, and kidney leukocytes. MiR-31 deficiency strikingly decreased systolic blood pressure and diastolic blood pressure and attenuated renal and vascular damage. MiR-31 deletion altered the accumulation of Treg cells and macrophages and expression of inflammatory cytokines in kidneys in Ang II-induced hypertensive mice. Ang II treatment reduced the levels of anti-inflammatory cytokines and increased proinflammatory cytokines in plasma that were blunted by the miR-31 deletion. Ppp6C (protein phosphatase 6c; a direct target of miR-31) specific deletion in Treg cells led to marked impairment of Treg cell induction, increased Ang II-induced blood pressure elevation, and organ damage in mice. In conclusion, we provided novel evidence of miR-31 as an emerging key posttranscriptional regulator of hypertension-associated immunosuppression through targeting ppp6C which is a critical regulator in the differentiation of Treg cells. This study offers new perspectives on miRNA-based therapeutic approaches.

Homoharringtonine deregulates MYC transcriptional expression by directly binding NF-κB repressing factor.

Homoharringtonine (HHT), a known protein synthesis inhibitor, has an anti-myeloid leukemia effect and potentiates the therapeutic efficacy of anthracycline/cytarabine induction regimens for acute myelogenous leukemia (AML) with favorable and intermediate prognoses, especially in the t(8;21) subtype. Here we provide evidence showing that HHT inhibits the activity of leukemia-initiating cells (Lin-/Sca-1-/c-kit+; LICs) in a t(8;21) murine leukemia model and exerts a down-regulating effect on MYC pathway genes in human t(8;21) leukemia cells (Kasumi-1). We discovered that NF-κB repressing factor (NKRF) is bound directly by HHT via the second double-strand RNA-binding motif (DSRM2) domain, which is the nuclear localization signal of NKRF. A series of deletion and mutagenesis experiments mapped HHT direct binding sites to K479 and C480 amino acids in the DSRM2 domain. HHT treatment shifts NKRF from the nucleus (including nucleoli) to the cytoplasm by occupying the DSRM2 domain, strengthens the p65-NKRF interaction, and interferes with p65-p50 complex formation, thereby attenuating the transactivation activity of p65 on the MYC gene. Moreover, HHT significantly decreases the expression of KIT, a frequently mutated and/or highly expressed gene in t(8;21) AML, in concert with MYC down-regulation. Our work thus identifies a mechanism of action of HHT that is different from, but acts in concert with, the known mode of action of this compound. These results justify further clinical testing of HHT in AML.

Arsenic circumvents the gefitinib resistance by binding to P62 and mediating autophagic degradation of EGFR in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is characterized by hyperexpression and/or gain-of-function mutations of the epidermal growth factor receptor (EGFR), resulting in an elevated overall kinase activity. Gefitinib is remarkably effective in patients with the L858R or ΔE746-A750-mutated of EGFR. However, drug resistance tends to develop because of the emergence of T790M mutation on EGFR. New strategies other than repressing kinase activity are thus required to treat NSCLC, thereby circumventing the resistance. In this study, arsenic trioxide (ATO) at 2 µM significantly inhibited the proliferation of the gefitinib-resistant NCI-H1975 cells of the EGFR L858R/T790M mutant compared with a modest inhibition in the gefitinib-sensitive HCC827 cells of ΔE746-A750 mutant and A549 cells of wild-type EGFR. Moreover, ATO significantly inhibited the overall kinase activity of EGFR primarily through quantitatively diminishing the EGFR in NCI-H1975 cells to an extent comparable with that reached by gefitinib in HCC827 cells. Furthermore, ATO promoted autophagic degradation of EGFR in NSCLC cells by directly binding to P62, which interacted with EGFR, preferentially the L858R/T790M mutant providing a plausible explanation for a more favorable effect of ATO on NCI-H1975 cells. Accordingly, the effect of ATO was further confirmed in the NSCLC xenograft mouse models. Our results reveal a new target for ATO with a unique molecular mechanism, i.e., ATO suppresses the overall catalytic potential of EGFR, significantly those with the L858R/T790M mutant in NCI-H1975 cells, through an autophagic degradation by interacting with P62. This study potentially offers an innovative therapeutic avenue for the NSCLC with L858R/T790M-mutated EGFR.

Transplantation of skin mesenchymal stem cells attenuated AngII-induced hypertension and vascular injury.

Skin mesenchymal stem cells (S-MSCs) revealed an important immunomodulatory activity to markedly suppress the formation of the atherosclerosis (AS) plaque by modulating macrophages, and also inhibit the development of experimental autoimmune encephalomyelitis (EAE) by regulating T helper 17 (Th17) cell differentiation. Macrophages and Th17 cells play important roles in hypertension. However, it remains unclear whether S-MSCs are capable of improving angiotensin (AngII)-induced hypertension by acting on inflammatory cells. Therefore, we studied a direct effect of S-MSC treatment on an AngII-induced hypertensive mouse model. Twenty-seven C57BL/6 (WT) mice were divided into three groups: Control group (WT-NC), AngII-infused group (WT-AngII), and S-MSC treatment group (WT-AngII + S-MSCs). In contrast to WT-AngII group, systolic blood pressure (SBP) and vascular damage were strikingly attenuated after tail-vein injection of S-MSCs. Numbers of Th17 cells in mouse peripheral blood of S-MSC treated group were significantly decreased, and IL-17 mRNA and protein levels were also reduced in the aorta and serum compared with WT-AngII group. Furthermore, macrophages in S-MSC treated group were switched to a regulatory profile characterized by a low ability to produce pro-inflammatory cytokine TNF-α and a high ability to produce anti-inflammatory cytokines Arg1 and IL-10. Mechanistically, we found that S-MSCs inhibited Th17 cell differentiation and induced M2 polarization. Moreover, we found proliferation and migration of S-MSCs were elevated, and expression of CXCR4, the receptor for Stromal derivated factor -1(SDF-1), was markedly increased in lipopolysaccharide (LPS)- stimulated S-MSCs. Given that SDF-1 expression was increased in the serum and aorta in AngII- induced hypertensive mice, the immunomodulatory effects exerted by S-MSCs involved the CXCR4/SDF-1 signaling. Collectively, our data demonstrated that S-MSCs attenuated AngII-induced hypertension by inhibiting Th17 cell differentiation and by modulating macrophage M2 polarization, suggesting that S-MSCs potentially have a role in stem cell based therapy for hypertension.

Roles of integrin ß3 cytoplasmic tail in bidirectional signal transduction in a trans-dominant inhibition model.

We evaluated the roles of calpain cleavage-related mutations of the integrin ß3 cytoplasmic tail in integrin αIIbß3 bidirectional signaling using a trans-dominant inhibition model. Chimeric Tac-ß3 proteins (i.e., Tac-ß3, Tac-ß3Δ741, Tac-ß3Δ747, Tac-ß3Δ754, Tac-ß3Δ759, and Tac-ß3ΔNITY) consisting of the extracellular and transmembrane domains of human IL-2 receptor (Tac) and the human integrin ß3 cytoplasmic domain were stably expressed in the 123 CHO cells harboring human glycoprotein Ib-IX and wild-type integrin αIIbß3. The different cells were assayed for stable adhesion and spreading on immobilized fibrinogen, and for binding soluble fibrinogen representing outside-in and inside-out signaling events, respectively. The chimeric protein Tac-ß3 inhibited, and Tac-ß3ΔNITY partially attenuated stable adhesion and spreading. Tac-ß3, Tac-ß3Δ759, Tac-ß3ΔNITY, and Tac-ß3Δ754, but not Tac-ß3Δ747 or Tac-ß3Δ741, impaired the soluble fibrinogen binding. Results indicated that the bidirectional signaling was significantly inhibited by Tac-ß3 and Tac-ß3ΔNITY, albeit to a much lesser extent. Moreover, only inside-out signaling was impaired in the 123/Tac-ß3Δ759 and 123/Tac-ß3Δ754 cells in contrast to an intact bidirectional signaling in the 123/Tac-ß3Δ747 and 123/Tac-ß3Δ741 cells. In conclusion, the calpain cleavage of integrin ß3 resulted in the regulatory effects on signaling by interrupting its interaction with cytoplasmic proteins rather than altering its conformation, and may thus regulate platelet function.

Skin-Derived Mesenchymal Stem Cells Alleviate Atherosclerosis via Modulating Macrophage Function.

UNLABELLED: Mesenchymal stem cells (MSCs) exhibit immunosuppressive efficacy and significantly inhibit the formation of the atherosclerosis (AS) plaque in apolipoprotein E-knockout (apoE(-/-)) mice. Of note, the largest lymphoid organ, the skin, provides a readily accessible and ideal source of tissue for the isolation of MSCs: skin-derived MSCs (S-MSCs). However, the effect and the mechanism of the therapeutic properties of S-MSCs in the progression of AS are unclear. We therefore investigated a direct effect of S-MSC treatment in the formation of atherosclerotic plaque in apoE(-/-) mice. Fifty apoE(-/-) mice were divided into four groups: the control group (AS), the S-MSC treatment group (S-MSC treatment), the nuclear factor-κB (NF-κB)(-/-)-S-MSC treatment group (KO-S-MSC treatment), and the additional S-MSC migration group. Brachiocephalic artery ultrasound biomicroscope (UBM) analysis showed that S-MSC treatment significantly reduced lesion size compared with the control groups (p < .01). Histological studies demonstrated that the plaque area of the mouse aortic arch was significantly decreased after S-MSC treatment. All alterations were dependent on NF-κB activation. After tail-vein injection, S-MSCs were capable of migrating to atherosclerotic plaque and selectively taking up residence near macrophages. S-MSC treatment reduced the release of the proinflammatory cytokine tumor necrosis factor (TNF)-α and increased the expression of the anti-inflammatory factor interleukin (IL)-10 in the atherosclerotic plaque, which was also dependent on NF-κB activation. In vitro, we found lipopolysaccharide (LPS) induced NF-κB-dependent expression of cyclooxygenase-2 (COX-2) in S-MSCs. Prostaglandin E2 (PGE2) expression was markedly increased after LPS-stimulated S-MSCs were cocultured with macrophages. LPS-stimulated macrophages produced less TNF-α/IL-1ß and more IL-10 when cultured with S-MSCs, and although both were dependent upon NF-κB, the release of IL-10 was diminished if the S-MSCs were pretreated with a COX-2 inhibitor or an EP2/EP4 antagonist. Our data demonstrated that S-MSCs inhibited the formation of the atherosclerotic plaque in apoE(-/-) mice by modulating the functionality of macrophages, suggesting that S-MSCs may potentially have a role in stem cell-based therapy for AS. SIGNIFICANCE: A combination of in vitro and in vivo experiments showed that skin-derived mesenchymal stem cells (S-MSCs) can attenuate the plaque size of atherosclerosis. This is probably because S-MSCs beneficially modulate the response of macrophages through an increased release of prostaglandin E2 acting on the EP2 and EP4 receptors of the macrophages, stimulating the production and release of the anti-inflammatory cytokine interleukin-10, and decreasing the production of proinflammatory cytokine tumor necrosis factor-α. S-MSCs inhibited the formation of the atherosclerotic plaque in apolipoprotein E-knockout mice by modulating the functionality of macrophages, and the suppressive property of S-MSCs is dependent on NF-κB signaling. This study provides direct evidence that S-MSCs have a potent immunosuppressive effect in the development of atherosclerosis in mice, suggesting that S-MSCs can easily be cultured and have similar function to bone marrow-derived MSCs, a promising cell source for stem cell-based therapies of atherosclerosis, and possibly also in transplantation.

Evaluation of targeting c-Src by the RGT-containing peptide as a novel antithrombotic strategy.

BACKGROUND: Interaction of integrin ß3 with c-Src plays critical roles in cellular signaling which is heavily implicated in platelet adhesion and aggregation, as well as in tumor cell proliferation and metastasis or in osteoclastic bone resorption. Selectively blocking integrin αIIbß3 outside-in signaling in platelets has been a focus of attention because of its effective antithrombotic potential together with a sufficient hemostatic capacity. The myristoylated RGT peptide has been shown to achieve this blockade by targeting the association of c-Src with the integrin ß3 tail, but the lack of key information regarding the mechanisms of action prevents this strategy from being further developed into practical antithrombotics. Therefore, in-depth knowledge of the precise mechanisms for RGT peptide in regulating platelet function is needed to establish the basis for a potential antithrombotic therapy by targeting c-Src. METHODS: The reduction-sensitive peptides were applied to rule out the membrane anchorage after cytoplasmic delivery. The c-Src activity was assayed at living cell or at protein levels to assess the direct effect of RGT targeting on c-Src. Thrombus formation under flow in the presence of cytoplasmic RGT peptide was observed by perfusing whole blood through the collagen-coated micro-chamber. RESULTS: The RGT peptide did not depend on the membrane anchorage to inhibit outside-in signaling in platelets. The myr-AC ~ CRGT peptide readily blocked agonist-induced c-Src activation by disrupting the Src/ß3 association and inhibited the RhoA activation and collagen-induced platelet aggregation in addition to the typical outside-in signaling events. The myr-AC ~ CRGT had no direct effect on the kinase activity of c-Src in living cells as evidenced by its inability to dissociate Csk from c-Src or to alter the phosphorylation level of c-Src Y(416) and Y(527), consistent results were also from in vitro kinase assays. Under flow conditions, the myr-AC~ CRGT peptide caused an inhibition of platelet thrombus formation predominantly at high shear rates. CONCLUSIONS: These findings provide novel insights into the molecular mechanisms by which the RGT peptide regulates integrin signaling and platelet function and reinforce the potential of the RGT peptide-induced disruption of Src/ß3 association as a druggable target that would finally enable in vivo and clinical studies using the structure-based small molecular mimetics.

PML/RARalpha fusion protein transactivates the tissue factor promoter through a GAGC-containing element without direct DNA association.

A severe coagulopathy is a life-threatening complication of acute promyelocytic leukemia (APL) and is ascribable mainly to the excessive levels of tissue factor (TF) in APL cells regulated in response to the promyelocytic leukemia/retinoic acid receptor alpha (PML/RARalpha) fusion protein. The underlying molecular mechanisms for this regulation remain ill-defined. With U937-PR9 cell lines stably expressing luciferase reporter gene under the control of different mutants of the TF promoter, both luciferase and ChIP data allowed the localization of the PML/RARalpha-responsive sequence in a previously undefined region of the TF promoter at position -230 to -242 devoid of known mammalian transcription factor binding sites. Within this sequence a GAGC motif (-235 to -238) was shown to be crucial because deletion or mutation of these nucleotides impaired both PML/RARalpha interaction and promoter transactivation. However, EMSA results showed that PML/RARalpha did not bind to DNA probes encompassing the -230 to -242 sequences, precluding a direct DNA association. Mutational experiments further suggest that the activator protein 1 (AP-1) sites of the TF promoter are dispensable for PML/RARalpha regulation. This study shows that PML/RARalpha transactivates the TF promoter through an indirect interaction with an element composed of a GAGC motif and the flanking nucleotides, independent of AP-1 binding.