Discovery of potent and specific CXCR3 antagonists

The optimization of a series of 8-aza-quinazolinone analogs for antagonist activity against the CXCR3 receptor is reported. Compounds were optimized to avoid the formation of active metabolites and time-dependent-inhibitors of CYP3A4. In addition, antagonists showed potent against CXCR3 activity in whole blood and optimized to avoid activity in the chromosomal aberration assay. Compound 25 was identified as having the optimal balance of CXCR3 activity and pharmacokinetic properties across multiple pre-clinical species, which are reported herein.     

Design and optimization of imidazole derivatives as potent CXCR3 antagonists

A series of imidazole derivatives have been designed and optimized for CXCR3 antagonism, pharmacokinetic properties, and reduced formation of glutathione conjugates. Our efforts led to the discovery of potent CXCR3 antagonists with good pharmacokinetic properties. These compounds are useful tools for in vivo studies of CXCR3 function.     

Osteopontin enhances the expression and activity of MMP-2 via the SDF-1/CXCR4 axis in hepatocellular carcinoma cell lines

Background and Aims

Osteopontin, SDF-1α, and MMP-2 are important secreted molecules involved in the pathophysiology of human hepatocellular carcinoma (HCC). This study investigates the effect of the SDF-1α/CXCR4 axis on expression and activity of MMP-2 induced by osteopontin.

Methods

The expression of CXCR4, SDF-1α, MMP-2 and their associated cellular signaling cascades, involving Akt and MAP Kinases, were determined by Western blotting. The activities of MMP-2 and MMP-9 were assayed by gel zymography. The role of the osteopontin receptors integrin α_vβ₃ and CD44v6 was evaluated using neutralizing antibodies. We also established CXCR4-deficient SMMC7721 cell lines by transfection with miRNA-CXCR4 plasmids and determined cell invasion activity in a transwell assay.

Results

In comparison with untreated cells, recombinant human osteopontin (rhOPN) up-regulated CXCR4, SDF-1α, and MMP-2 expression about 5-, 4-, and 6-fold on the protein levels through binding to integrin α_vβ₃ and CD44v6 in hepatocellular carcinoma cells (SMMC7721 and HepG2). Inhibition of the SDF-1α/CXCR4 axis down-regulated the rhOPN-induced MMP-2 expression and activity. rhOPN also activated Akt, p38 and JNK. Down-regulation of CXCR4 decreased the rhOPN-induced invasion in SMMC7721 cells.

Conclusion

These results indicate that rhOPN up-regulates MMP-2 through the SDF-1α/CXCR4 axis, mediated by binding to integrin α_vβ₃ and CD44v6 and activating the PI-3K/Akt and JNK pathways in HepG2 and SMMC7721 cells. Therefore, the osteopontin-SDF-1α/CXCR4-MMP-2 system may be a new therapeutic target for treating HCC progression.     

High frequency of CD4+ CXCR5+ TFH cells in patients with immune-active chronic hepatitis B

Background

T follicular helper (TFH) cells are a special subpopulation of T helper cells and can regulate humoral immune responses. This study examined whether the frequency of CD4⁺CXCR5⁺ TFH cells could be associated with active immunity in chronic hepatitis B (CHB) patients.

Methodology and Findings

The frequencies of peripheral blood CD4⁺CXCR5⁺ TFH cells, inducible T cell costimulator (ICOS), and/or programmed death 1 (PD-1) positive CD4⁺CXCR5⁺ TFH cells in immune-active (IA), immune-tolerant (IT) CHB, and healthy controls (HC) were characterized by flow cytometry analysis. The effect of adevofir dipivoxil treatment on the frequency of CD4⁺CXCR5⁺ TFH cells, the concentrations of serum IL-2, IFN-γ, TNF-α, IL-4, IL-6, IL-10, IL-21, ALT, AST, HBsAg, HBsAb, HBeAg, HBeAb and HBV loads in IA patients were determined. The potential association of the frequency of CD4⁺CXCR5⁺ TFH cells with clinical measures was analyzed. In addition, the frequency of splenic and liver CD4⁺CXCR5⁺ TFH cells in HBV-transgenic mice was examined. We found that the frequency of CD4⁺CXCR5⁺ TFH cells in IA patients was significantly higher than that of IT patients and HC, and the percentages of CD4⁺CXCR5⁺ TFH in IA patients were positively correlated with AST. Furthermore, the percentages of ICOS⁺, PD-1⁺, and ICOS⁺PD-1⁺ in CD4⁺CXCR5⁺ TFH cells in CHB patients were significantly higher than that of HC. Treatment with adefovir dipivoxil reduced the frequency of CD4⁺CXCR5⁺ TFH, PD-1⁺CD4⁺CXCR5⁺ TFH cells and the concentrations of HBsAg and HBeAg, but increased the concentrations of HBsAb, HBeAb, IL-2 and IFN-γ in IA patients. Moreover, the frequency of splenic and liver CD4⁺CXCR5⁺ TFH cells in HBV-transgenic mice was higher than that of wild-type controls.

Conclusions

These data indicate that CD4⁺CXCR5⁺ TFH cells may participate in the HBV-related immune responses and that high frequency of CD4⁺CXCR5⁺ TFH cells may be a biomarker for the evaluation of active immune stage of CHB patients.     

X4 Human immunodeficiency virus type 1 gp120 promotes human hepatic stellate cell activation and collagen I expression through interactions with CXCR4

Background & Aims

Patients coinfected with HIV-1 and HCV develop more rapid liver fibrosis than patients monoinfected with HCV. HIV RNA levels correlate with fibrosis progression implicating HIV directly in the fibrotic process. While activated hepatic stellate cells (HSCs) express the 2 major HIV chemokine coreceptors, CXCR4 and CCR5, little is known about the pro-fibrogenic effects of the HIV-1 envelope protein, gp120, on HSCs. We therefore examined the in vitro impact of X4 gp120 on HSC activation, collagen I expression, and underlying signaling pathways and examined the in vivo expression of gp120 in HIV/HCV coinfected livers.

Methods

Primary human HSCs and LX-2 cells, a human HSC line, were challenged with X4 gp120 and expression of fibrogenic markers assessed by qRT-PCR and Western blot +/− either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Downstream intracellular signaling pathways were evaluated with Western blot and pre-treatment with specific pathway inhibitors. Gp120 immunostaining was performed on HIV/HCV coinfected liver biopsies.

Results

X4 gp 120 significantly increased expression of alpha-smooth muscle actin (a-SMA) and collagen I in HSCs which was blocked by pre-incubation with either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Furthermore, X4 gp120 promoted Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and pretreatment with an ERK inhibitor attenuated HSC activation and collagen I expression. Sinusoidal staining for gp120 was evident in HIV/HCV coinfected livers.

Conclusions

X4 HIV-1 gp120 is pro-fibrogenic through its interactions with CXCR4 on activated HSCs. The availability of small molecule inhibitors to CXCR4 make this a potential anti-fibrotic target in HIV/HCV coinfected patients.     

Correlation of circulating CD133+ progenitor subclasses with a mild phenotype in Duchenne muscular dystrophy patients

Background

Various prognostic serum and cellular markers have been identified for many diseases, such as cardiovascular diseases and tumor pathologies. Here we assessed whether the levels of certain stem cells may predict the progression of Duchenne muscular dystrophy (DMD).

Methods and Findings

The levels of several subpopulations of circulating stem cells expressing the CD133 antigen were determined by flow cytometry in 70 DMD patients. The correlation between the levels and clinical status was assessed by statistical analysis. The median (±SD) age of the population was 10.66±3.81 (range 3 to 20 years). The levels of CD133+CXCR4+CD34- stem cells were significantly higher in DMD patients compared to healthy controls (mean±standard deviation: 17.38±1.38 vs. 11.0±1.70; P = 0.03) with a tendency towards decreased levels in older patients. Moreover, the levels of this subpopulation of cells correlated with the clinical condition. In a subgroup of 19 DMD patients after 24 months of follow-up, increased levels of CD133+CXCR4+CD34- cells was shown to be associated with a phenotype characterised by slower disease progression. The circulating CD133+CXCR4+CD34- cells in patients from different ages did not exhibit significant differences in their myogenic and endothelial in vitro differentiation capacity.

Conclusions

Our results suggest that levels of CD133+CXCR4+CD34- could function as a new prognostic clinical marker for the progression of DMD.     

Thermal stability of the human immunodeficiency virus type 1 (HIV-1) receptors, CD4 and CXCR4, reconstituted in proteoliposomes

Background

The entry of human immunodeficiency virus (HIV-1) into host cells involves the interaction of the viral exterior envelope glycoprotein, gp120, and receptors on the target cell. The HIV-1 receptors are CD4 and one of two chemokine receptors, CCR5 or CXCR4.

Methodology/Principal Findings

We created proteoliposomes that contain CD4, the primary HIV-1 receptor, and one of the coreceptors, CXCR4. Antibodies against CD4 and CXCR4 specifically bound the proteoliposomes. CXCL12, the natural ligand for CXCR4, and the small-molecule CXCR4 antagonist, AMD3100, bound the proteoliposomes with affinities close to those associated with the binding of these molecules to cells expressing CXCR4 and CD4. The HIV-1 gp120 exterior envelope glycoprotein bound tightly to proteoliposomes expressing only CD4 and, in the presence of soluble CD4, bound weakly to proteoliposomes expressing only CXCR4. The thermal stability of CD4 and CXCR4 inserted into liposomes was examined. Thermal denaturation of CXCR4 followed second-order kinetics, with an activation energy (E_a) of 269 kJ/mol (64.3 kcal/mol) and an inactivation temperature (T_i) of 56°C. Thermal inactivation of CD4 exhibited a reaction order of 1.3, an E_a of 278 kJ/mol (66.5 kcal/mol), and a T_i of 52.2°C. The second-order denaturation kinetics of CXCR4 is unusual among G protein-coupled receptors, and may result from dimeric interactions between CXCR4 molecules.

Conclusions/Significance

Our studies with proteoliposomes containing the native HIV-1 receptors allowed an examination of the binding of biologically important ligands and revealed the higher-order denaturation kinetics of these receptors. CD4/CXCR4-proteoliposomes may be useful for the study of virus-target cell interactions and for the identification of inhibitors.     

The Maturational Refolding of the β-Hairpin Motif of Equine Infectious Anemia Virus Capsid Protein Extends Its Helix α1 at Capsid Assembly Locus

A retroviral capsid (CA) protein consists of two helical domains, CA^N and CA^C, which drive hexamer and dimer formations, respectively, to form a capsid lattice. The N-terminal 13 residues of CA refold to a β-hairpin motif upon processing from its precursor polyprotein Gag. The β-hairpin is essential for correct CA assembly but unexpectedly it is not within any CA oligomeric interfaces. To understand the β-hairpin function we studied the full-length CA protein from equine infectious anemia virus (EIAV), a lentivirus sharing the same cone-shaped capsid core as HIV-1. Solution NMR spectroscopy is perfectly suited to study EIAV-CA that dimerizes weaker than HIV-1-CA. Comparison between the wild-type (wt) EIAV-CA and a variant lacking the β-hairpin structure demonstrated that folding of the β-hairpin specifically extended the N terminus of helix α1 from Tyr²⁰ to Pro¹⁷. This coil to helix transition involves the conserved sequence of Thr¹⁶-Pro¹⁷-Arg¹⁸ (Ser¹⁶-Pro¹⁷-Arg¹⁸ in HIV-1-CA). The extended region of helix α1 constituted an expanded EIAV-CA^N oligomeric interface and overlapped with the HIV-1-CA hexamer-core residue Arg¹⁸, helical in structure and pivotal in assembly. Therefore we propose the function of the maturational refolding of the β-hairpin in CA assembly is to extend helix α1 at the N terminus to enhance the CA^N oligomerization along the capsid assembly core interface. In addition, NMR resonance line broadening indicated the presence of micro-millisecond exchange kinetics due to the EIAV-CA^N domain oligomerization, independent to the faster EIAV-CA^C domain dimerization.     

T134, a Small-Molecule CXCR4 Inhibitor, Has No Cross-Drug Resistance with AMD3100, a CXCR4 Antagonist with a Different Structure

T22, an analog of polyphemusin II (18 amino acid residues), was found to block T-tropic human immunodeficiency virus type 1 (HIV-1) entry into target cells as a CXCR4 inhibitor. We synthesized T134, a small analog (14 amino acid residues) of T22 with reduced positive charges. T134 exhibited highly potent activity and significantly less cytotoxicity in comparison to that of T22. T134 prevents the anti-CXCR4 monoclonal antibody from binding to peripheral blood mononuclear cells but has no effect on the binding of anti-CCR5 monoclonal antibodies. Since T134 inhibits the binding of stromal cell-derived factor-1 (SDF-1) to MT-4 cells, it seems that T134 prevents HIV-1 entry by binding to CXCR4. The bicyclam AMD3100 has also been shown to block HIV-1 entry via CXCR4 but not via CCR5. Both T134 and AMD3100 are CXCR4 antagonists and low-molecular-weight compounds but have different structures. Our results indicate that T134 is active against wild-type T-tropic HIV-1 strains and against AMD3100-resistant strains.     

Probing the opioid receptor complex with (+)-trans-SUPERFIT. II. Evidence that mu ligands are noncompetitive inhibitors of the delta cx opioid peptide binding site.

Previous studies delineated two classes of δ binding sites; a δ binding site not associated with the opioid receptor complex, termed the δ_ncx site, and a δ site associated with the opioid receptor complex, termed the δ_cx site. The δ_ncx site has high affinity for [ -Pen², -Pen⁵]enkephalin, and is synonymous with what is now identified as the δ₁ binding site. Pretreatment of membranes with the δ-selective acylating agents FIT, or (+)-trans-SUPERFIT, deplete membranes of the δ_ncx binding site, which permits the selective labeling of the δ_cx binding site with [³H][ -Ala²,Leu⁵]enkephalin. The present study compared the properties of the δ_cx binding site present in brain membranes pretreated with (+)-trans-SUPERFIT with the properties of the δ_cx site present in untreated membranes. The major findings are: 1) pretreatment of membranes with (+)-trans-SUPERFIT decreased the IC₅₀ values of δ-preferring drugs, and increased the IC₅₀ values of μ-preferring drugs, for the δ_cx binding site; 2) the degree of δ selectivity was highly correlated with the magnitude of the (+)-trans-SUPERFIT-induced shift in the IC₅₀ values; 3) the ligand-selectivity patterns of the μ and δ_cx sites present in (+)-trans-SUPERFIT-pretreated membranes were poorly correlated; 4) whereas μ-preferring drugs were noncompetitive inhibitors of [³H][ -Ala²,Leu⁵]enkephalin binding to the δ_cx site, δ-preferring drugs were competitive inhibitors. Viewed collectively, these data support the hypothesis that the μ and δ_cx binding sites are distinct, provide additional evidence for δ receptor heterogeneity, and suggest that ( (+)-trans-SUPERFIT-pretreated membranes will provide a useful preparation for studying the δ_cx binding site.     

The CXCR3(+)CD56Bright phenotype characterizes a distinct NK cell subset with anti-fibrotic potential that shows dys-regulated activity in hepatitis C

Background

In mouse models, natural killer (NK) cells have been shown to exert anti-fibrotic activity via killing of activated hepatic stellate cells (HSC). Chemokines and chemokine receptors critically modulate hepatic recruitment of NK cells. In hepatitis C, the chemokine receptor CXCR3 and its ligands have been shown to be associated with stage of fibrosis suggesting a role of these chemokines in HCV associated liver damage by yet incompletely understood mechanisms. Here, we analyzed phenotype and function of CXCR3 expressing NK cells in chronic hepatitis C.

Methods

Circulating NK cells from HCV-infected patients (n = 57) and healthy controls (n = 27) were analyzed with respect to CXCR3 and co-expression of different maturation markers. Degranulation and interferon-γ secretion of CXCR3(+) and CXCR3(−) NK cell subsets were studied after co-incubation with primary human hepatic stellate cells (HSC). In addition, intra-hepatic frequency of CXCR3(+) NK cells was correlated with stage of liver fibrosis (n = 15).

Results

We show that distinct NK cell subsets can be distinguished based on CXCR3 surface expression. In healthy controls CXCR3(+)CD56Bright NK cells displayed strongest activity against HSC. Chronic hepatitis C was associated with a significantly increased frequency of CXCR3(+)CD56Bright NK cells which showed impaired degranulation and impaired IFN-γ secretion in response to HSC. Of note, we observed intra-hepatic accumulation of this NK cell subset in advanced stages of liver fibrosis.

Conclusion

We show that distinct NK cell subsets can be distinguished based on CXCR3 surface expression. Intra-hepatic accumulation of the functionally impaired CXCR3(+)CD56Bright NK cell subset might be involved in HCV-induced liver fibrosis.     

Conformational study of a highly specific CXCR4 inhibitor, T140, disclosing the close proximity of its intrinsic pharmacophores associated with strong anti-HIV activity

We report the solution structure of T140, a truncated polyphemusin peptide analogue that efficiently inhibits infection of target cells by T-cell line-tropic strains of HIV-1 through its specific binding to a chemokine receptor, CXCR4. Nuclear magnetic resonance analysis and molecular dynamic calculations revealed that T140 has a rigidly structured conformation constituted by an antiparallel beta-sheet and a type II' beta-turn. A protuberance is formed on one side of the beta-sheet by the side-chain functional groups of the three amino acid residues (L-3-(2-naphthyl)alanine, Tyr5 and Arg14), each of which is indispensable for strong anti-HIV activity. These findings provide a rationale to dissect the structural basis for the ability of this compound to block the interaction between CXCR4 and envelope glycoproteins from T-tropic strains of HIV-1.     

Coreceptor and cytokine concentrations may not explain differences in disease progression observed in HIV-1 clade A and D infected Ugandans

Background

The use of cellular coreceptors and modulation of cytokine concentrations by HIV to establish a productive infection is well documented. However, it is unknown whether the expression of these proteins affects the course of HIV clade A and D disease, reported to have different progression rates.

Methodology/Principal Findings

We investigated whether the number of CD4⁺ T-cells expressing CCR5 or CXCR4, the density of these coreceptors and concentrations of specific immune proteins linked to HIV pathogenesis vary between individuals infected with HIV clade A or D. We undertook additional analyses stratifying participants by early (CD4>500 cells/µl) or late (CD4<200 cells/µl) disease stage. Whole blood samples were taken from 50 HIV-1 infected individuals drawn from cohorts in rural south-west Uganda. Late stage participants had less than half the number of CD4⁺/CCR5⁺ T-cells (p = 0.0113) and 5.6 times fewer CD4⁺/CXCR4⁺ cells (p<0.0001) than early stage participants. There was also a statistically significant difference in the density of CXCR4 on CD4⁺ cells between clade A and D infected early stage participants (142 [A] vs 84 [D]; p = 0.0146). Across all participants we observed significantly higher concentration of Th₁ cytokines compared to Th₂ (66.4 vs 23.8 pg/ml; p<0.0001). Plasma concentrations of IFNγ and IL-2 were 1.8 and 2.4 fold lower respectively in Late-D infected participants compared to Late-A participants. MIP-1β levels also decreased from 118.0 pg/ml to 47.1 pg/ml (p = 0.0396) as HIV disease progressed.

Conclusions/Significance

We observed specific alterations in the abundance of CD4⁺/CCR5⁺ and CD4⁺/CXCR4⁺ T-cells, and concentrations of immune proteins across different HIV clades and as infection progresses. Our results suggest that these changes are unlikely to explain the observed differences in disease progression between subtype A and D infections. However, our observations further the understanding of the natural progression of non-clade B HIV infection and how the virus adapts to exploit the host environment.     

Optimisation of pharmacokinetic properties in a neutral series of 11β-HSD1 inhibitors

11β-HSD1 is increasingly seen as an attractive target for the treatment of type II diabetes and other elements of the metabolic syndrome. In this program of work we describe how a series of neutral 2-thioalkyl-pyridine 11β-HSD1 inhibitors were optimized in terms of their pharmacokinetic properties to give compounds with excellent bioavailability in both rat and dog through a core change to pyrimidine. A potential reactive metabolite issue with 4-thioalkyl-pyrimidines was circumvented by a switch from sulfur to carbon substitution.     

Attenuation of osteoarthritis via blockade of the SDF-1/CXCR4 signaling pathway

Introduction

This study was performed to evaluate the attenuation of osteoarthritic (OA) pathogenesis via disruption of the stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) signaling with AMD3100 in a guinea pig OA model.

Methods

OA chondrocytes and cartilage explants were incubated with SDF-1, siRNA CXCR4, or anti-CXCR4 antibody before treatment with SDF-1. Matrix metalloproteases (MMPs) mRNA and protein levels were measured with real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The 35 9-month-old male Hartley guinea pigs (0.88 kg ± 0.21 kg) were divided into three groups: AMD-treated group (n = 13); OA group (n = 11); and sham group (n = 11). At 3 months after treatment, knee joints, synovial fluid, and serum were collected for histologic and biochemical analysis. The severity of cartilage damage was assessed by using the modified Mankin score. The levels of SDF-1, glycosaminoglycans (GAGs), MMP-1, MMP-13, and interleukin-1 (IL-1β) were quantified with ELISA.

Results

SDF-1 infiltrated cartilage and decreased proteoglycan staining. Increased glycosaminoglycans and MMP-13 activity were found in the culture media in response to SDF-1 treatment. Disrupting the interaction between SDF-1 and CXCR4 with siRNA CXCR4 or CXCR4 antibody attenuated the effect of SDF-1. Safranin-O staining revealed less cartilage damage in the AMD3100-treated animals with the lowest Mankin score compared with the control animals. The levels of SDF-1, GAG, MMP1, MMP-13, and IL-1β were much lower in the synovial fluid of the AMD3100 group than in that of control group.

Conclusions

The binding of SDF-1 to CXCR4 induces OA cartilage degeneration. The catabolic processes can be disrupted by pharmacologic blockade of SDF-1/CXCR4 signaling. Together, these findings raise the possibility that disruption of the SDF-1/CXCR4 signaling can be used as a therapeutic approach to attenuate cartilage degeneration.     

Interleukin-8 is activated in patients with chronic liver diseases and associated with hepatic macrophage accumulation in human liver fibrosis

Background

Interleukin-8 (IL-8, CXCL8) is a potent chemoattractant for neutrophils and contributes to acute liver inflammation. Much less is known about IL-8 in chronic liver diseases (CLD), but elevated levels were reported from alcoholic and hepatitis C-related CLD. We investigated the regulation of IL-8, its receptors CXCR1 and CXCR2 and possible IL-8 responding cells in CLD patients.

Methodology

Serum IL-8 levels were measured in CLD patients (n = 200) and healthy controls (n = 141). Intrahepatic IL-8, CXCR1 and CXCR2 gene expression was quantified from liver samples (n = 41), alongside immunohistochemical neutrophil (MPO) and macrophage (CD68) stainings. CXCR1 and CXCR2 expression was analyzed on purified monocytes from patients (n = 111) and controls (n = 31). In vitro analyses explored IL-8 secretion by different leukocyte subsets.

Principal Findings

IL-8 serum levels were significantly increased in CLD patients, especially in end-stage cirrhosis. Interestingly, patients with cholestatic diseases exhibited highest IL-8 serum concentrations. IL-8 correlated with liver function, inflammatory cytokines and non-invasive fibrosis markers. Intrahepatically, IL-8 and CXCR1 expression were strongly up-regulated. However, intrahepatic IL-8 could only be associated to neutrophil infiltration in patients with primary biliary cirrhosis (PBC). In non-cholestatic cirrhosis, increased IL-8 and CXCR1 levels were associated with hepatic macrophage accumulation. In line, CXCR1, but not CXCR2 or CXCR3, expression was increased on circulating monocytes from cirrhotic patients. Moreover, monocyte-derived macrophages from CLD patients, especially the non-classical CD16⁺ subtype, displayed enhanced IL-8 secretion in vitro.

Conclusions

IL-8 is strongly activated in CLD, thus likely contributing to hepatic inflammation. Our study suggests a novel role of IL-8 for recruitment and activation of hepatic macrophages via CXCR1 in human liver cirrhosis.     

Hypoxic preconditioning improves survival of cardiac progenitor cells: role of stromal cell derived factor-1α-CXCR4 axis

Background

Cardiac progenitor cells (CPCs) have been shown to be suitable in stem cell therapy for resurrecting damaged myocardium, but poor retention of transplanted cells in the ischemic myocardium causes ineffective cell therapy. Hypoxic preconditioning of cells can increase the expression of CXCR4 and pro-survival genes to promote better cell survival; however, it is unknown whether hypoxia preconditioning will influence the survival and retention of CPCs via the SDF-1α/CXCR4 axis.

Methods and Results

CPCs were isolated from adult mouse hearts and purified by magnetic activated cell sorting using c-kit magnetic beads. These cells were cultured at various times in either normoxic or hypoxic conditions, and cell survival was analyzed using flow cytometry and the expression of hypoxia-inducible factor-1α (HIF-1α), CXCR4, phosphorylated Akt and Bcl-2 were measured by Western blot. Results showed that the expression of pro-survival genes significantly increased after hypoxia treatment, especially in cells cultured in hypoxic conditions for six hours. Upon completion of hypoxia preconditioning from c-kit+ CPCs for six hours, the anti-apoptosis, migration and cardiac repair potential were evaluated. Results showed a significant enhancement in anti-apoptosis and migration in vitro, and better survival and cardiac function after being transplanted into acute myocardial infarction (MI) mice in vivo. The beneficial effects induced by hypoxia preconditioning of c-kit+ CPCs could largely be blocked by the addition of CXCR4 selective antagonist AMD3100.

Conclusions

Hypoxic preconditioning may improve the survival and retention of c-kit+ CPCs in the ischemic heart tissue through activating the SDF-1α/CXCR4 axis and the downstream anti-apoptosis pathway. Strategies targeting this aspect may enhance the effectiveness of cell-based cardiac regenerative therapy.     

Interaction of polyphemusin I and structural analogs with bacterial membranes, lipopolysaccharide, and lipid monolayers

Three structural variants (PV5, PV7, and PV8) of the horseshoe crab cationic antimicrobial peptide polyphemusin I were designed with improved amphipathic profiles. Circular dichroism spectroscopy analysis indicated that in phosphate buffer polyphemusin I, PV7, and PV8 displayed the spectrum of a type II beta-turn-rich structure, but, like polyphemusin I, all three variants adopted a typical beta-sheet structure in an anionic lipid environment. Both polyphemusin I and variants were potent broad spectrum antimicrobials that were clearly bactericidal at their minimal inhibitory concentrations. The variants were moderately less active in vitro but more effective in animal models. Moreover, these variants exhibited delayed bacterial killing, whereas polyphemusin I killed Escherichia coli UB1005 within 5 min at 2.5 microg/mL. All the peptides showed similar abilities to bind to bacterial lipopolysaccharide (LPS) and permeabilize bacterial outer membranes. Consistent with this was the observation that all peptides significantly inhibited cytokine production by LPS-stimulated macrophages and penetrated polyanionic LPS monolayers to similar extents. None of the peptides had affinity for neutral lipids as evident from both tryptophan fluorescence spectroscopy and Langmuir monolayer analysis. As compared to polyphemusin I, all variants showed reduced ability to interact with anionic lipids, and the hemolytic activity of the variants was decreased by 2-4-fold. In contrast, polyphemusin I efficiently depolarized the cytoplasmic membrane of E. coli, as assessed using a membrane potential sensitive fluorescent dye 3,3-dipropylthiacarbocyanine (diSC(3)5) assay, but the variants showed a substantially delayed and decreased depolarizing ability. The coincident assessment of cell viability indicated that depolarization of the bacterial cytoplasmic membrane potential by polyphemusin I occurred prior to lethal damage to cells. Our data suggest that increase of amphipathicity of beta-sheet polyphemusin I generally resulted in variants with decreased activity for membranes. Interestingly, all variants showed an improved ability to protect mice both against infection by Pseudomonas aeruginosa and from endotoxaemia.     

Structural relationships in the lysozyme superfamily: significant evidence for glycoside hydrolase signature motifs

Background

Chitin is a polysaccharide that forms the hard, outer shell of arthropods and the cell walls of fungi and some algae. Peptidoglycan is a polymer of sugars and amino acids constituting the cell walls of most bacteria. Enzymes that are able to hydrolyze these cell membrane polymers generally play important roles for protecting plants and animals against infection with insects and pathogens. A particular group of such glycoside hydrolase enzymes share some common features in their three-dimensional structure and in their molecular mechanism, forming the lysozyme superfamily.

Results

Besides having a similar fold, all known catalytic domains of glycoside hydrolase proteins of lysozyme superfamily (families and subfamilies GH19, GH22, GH23, GH24 and GH46) share in common two structural elements: the central helix of the all-α domain, which invariably contains the catalytic glutamate residue acting as general-acid catalyst, and a β-hairpin pointed towards the substrate binding cleft. The invariant β-hairpin structure is interestingly found to display the highest amino acid conservation in aligned sequences of a given family, thereby allowing to define signature motifs for each GH family. Most of such signature motifs are found to have promising performances for searching sequence databases. Our structural analysis further indicates that the GH motifs participate in enzymatic catalysis essentially by containing the catalytic water positioning residue of inverting mechanism.

Conclusions

The seven families and subfamilies of the lysozyme superfamily all have in common a β-hairpin structure which displays a family-specific sequence motif. These GH β-hairpin motifs contain potentially important residues for the catalytic activity, thereby suggesting the participation of the GH motif to catalysis and also revealing a common catalytic scheme utilized by enzymes of the lysozyme superfamily.