Discovery of AD258 as a Sigma Receptor Ligand with Potent Antiallodynic Activity.

The design and synthesis of a series of 2,7-diazaspiro[4.4]nonane derivatives as potent sigma receptor (SR) ligands, associated with analgesic activity, are the focus of this work. In this study, affinities at S1R and S2R were measured, and molecular modeling studies were performed to investigate the binding pose characteristics. The most promising compounds were subjected to in vitro toxicity testing and subsequently screened for in vivo analgesic properties. Compound 9d (AD258) exhibited negligible in vitro cellular toxicity and a high binding affinity to both SRs (KiS1R = 3.5 nM, KiS2R = 2.6 nM), but not for other pain-related targets, and exerted high potency in a model of capsaicin-induced allodynia, reaching the maximum antiallodynic effect at very low doses (0.6-1.25 mg/kg). Functional activity experiments showed that S1R antagonism is needed for the effects of 9d and that it did not induce motor impairment. In addition, 9d exhibited a favorable pharmacokinetic profile.

Modulation of ocular surface desiccation in a murine model by histatin-5 application.

PURPOSE: To determine the efficacy of Histatin-5 (Hst5) peptide treatment in ameliorating dry eye disease (DED) phenotype in an in-vivo mouse model of scopolamine and desiccating stress (SDS) dry eye. METHODS: SDS was induced in female C57BL/6 mice by subcutaneous injections of scopolamine hydrobromide and exposure to low relative humidity and forced air draft for five days. Mouse eyes were topically treated with synthetic Hst5 peptide or balanced salt solution (BSS) twice a day for four days. Control mice were not exposed to SDS induction and did not receive any treatments. Oregon green dextran (OGD) staining was used to evaluate corneal permeability. Histologically, staining with periodic acid schiff (PAS), immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), were used to quantify the number of goblet cells (GC), CD45+ immune cells and apoptotic cells respectively in formalin fixed paraffin embedded (FFPE) mouse whole eye sections. RESULTS: Compared to treatment with BSS, Hst5 treatment significantly lowered corneal epithelial permeability, prevented conjunctival epithelial GC loss, decreased conjunctival CD45+ immune cell infiltration and reduced conjunctival epithelial cell apoptosis. CONCLUSIONS: Hst5 peptide topical treatment significantly improves the clinical parameters observed in SDS experimental model of DED. This is the first report of the efficacy of Hst5 treatment of dry eye phenotype, and potential novel treatment for DED in the clinic. Hst5 represents a new class of efficacious therapeutic agents, demonstrating pro-epithelial and anti-inflammatory activities at the ocular surface.

Histatin-1 Attenuates LPS-Induced Inflammatory Signaling in RAW264.7 Macrophages.

Macrophages play a critical role in the inflammatory response to environmental triggers, such as lipopolysaccharide (LPS). Inflammatory signaling through macrophages and the innate immune system are increasingly recognized as important contributors to multiple acute and chronic disease processes. Nitric oxide (NO) is a free radical that plays an important role in immune and inflammatory responses as an important intercellular messenger. In addition, NO has an important role in inflammatory responses in mucosal environments such as the ocular surface. Histatin peptides are well-established antimicrobial and wound healing agents. These peptides are important in multiple biological systems, playing roles in responses to the environment and immunomodulation. Given the importance of macrophages in responses to environmental triggers and pathogens, we investigated the effect of histatin-1 (Hst1) on LPS-induced inflammatory responses and the underlying molecular mechanisms in RAW264.7 (RAW) macrophages. LPS-induced inflammatory signaling, NO production and cytokine production in macrophages were tested in response to treatment with Hst1. Hst1 application significantly reduced LPS-induced NO production, inflammatory cytokine production, and inflammatory signaling through the JNK and NF-kB pathways in RAW cells. These results demonstrate that Hst1 can inhibit LPS-induced inflammatory mediator production and MAPK signaling pathways in macrophages.

Histatin-1 is an endogenous ligand of the sigma-2 receptor.

The Sigma-2 receptor (S2R) (a.k.a TMEM97) is an important endoplasmic reticular protein involved in cancer, cholesterol processing, cell migration, and neurodegenerative diseases, including Niemann-Pick Type C. While several S2R pharmacologic agents have been discovered, its recent (2017) cloning has limited biological investigation, and no endogenous ligands of the S2R are known. Histatins are a family of endogenous antimicrobial peptides that have numerous important effects in multiple biological systems, including antifungal, antibacterial, cancer pathogenesis, immunomodulation, and wound healing. Histatin-1 (Hst1) has important roles in epithelial wound healing and cell migration, and is the primary wound healing agent in saliva. Little is understood about the downstream machinery that underpins the effects of histatins, and no mammalian receptor is known to date. In this study, we show, using biophysical methods and functional assays, that Hst1 is an endogenous ligand for S2R and that S2R is a mammalian receptor for Hst1.

Electrothermal soft manipulator enabling safe transport and handling of thin cell/tissue sheets and bioelectronic devices.

"Living" cell sheets or bioelectronic chips have great potentials to improve the quality of diagnostics and therapies. However, handling these thin and delicate materials remains a grand challenge because the external force applied for gripping and releasing can easily deform or damage the materials. This study presents a soft manipulator that can manipulate and transport cell/tissue sheets and ultrathin wearable biosensing devices seamlessly by recapitulating how a cephalopod's suction cup works. The soft manipulator consists of an ultrafast thermo-responsive, microchanneled hydrogel layer with tissue-like softness and an electric heater layer. The electric current to the manipulator drives microchannels of the gel to shrink/expand and results in a pressure change through the microchannels. The manipulator can lift/detach an object within 10 s and can be used repeatedly over 50 times. This soft manipulator would be highly useful for safe and reliable assembly and implantation of therapeutic cell/tissue sheets and biosensing devices.

Wound Healing Properties of Histatin-5 and Identification of a Functional Domain Required for Histatin-5-Induced Cell Migration.

Histatin peptides are endogenous anti-microbial peptides that were originally discovered in the saliva. Aside from their broad anti-microbial properties, these peptides play an important role in multiple biological systems. Different members of this family are thought to have relative specializations, with histatin-5 originally being thought to have mostly anti-fungal properties, and histatin-1 having strong wound healing properties. In this report, we describe the robust wound healing properties of histatin-5 and elucidate a functional domain, which is necessary and sufficient for promoting wound healing. We demonstrate these findings in multiple different cell types in vitro and with a standardized murine corneal wound healing model. Discovery of this wound healing domain and description of this functional role of histatin-5 will support developing therapies.

Presence of Histatin-1 in Human Tears and Association with Aqueous Deficient Dry Eye Diagnosis: A Preliminary Study.

The aims of this study were to determine if histatin-1 (H1) is present in normal human tears and whether tear levels of H1 varied between normal patients and those with aqueous deficient dry eye disease (ADDE). Patient samples were obtained from 11 normal patients and 11 severe ADDE patients. Relevant patient characteristics, including age, sex, and dry eye disease (DED) diagnostic parameters were collected. Multiple qualitative and quantitative methods were used to compare the concentration of H1 between patient groups. Mixed linear modeling was used to compare H1 levels between groups, and diagnostic performance was assessed using the receiver-operator-characteristic (ROC). ADDE patients had significantly lower H1 concentrations (85.9 ± 63.7 ng/ml) than the normal group (891.6 ± 196.5 ng/ml) (p < 0.001), while controlling for age and sex. ROC analysis indicated that H1 concentration is potentially a biomarker for ADDE (area under curve = 0.96). Reclassification of patients by DED parameters including, Ocular Surface Disease Index (OSDI) (≤13, >13) and Schirmer I (without anesthesia) (<10 mm, ≥10 mm) showed significant differences in H1 level (OSDI, p = 0.004) and Schirmer I ((p = 0.010). In conclusion, this is the first preliminary report of the presence of H1 in human tears. H1 concentrations are lower in ADDE patients and H1 may have diagnostic potential in evaluation ADDE patients.

microRNA-219 Reduces Viral Load and Pathologic Changes in Theiler's Virus-Induced Demyelinating Disease.

Analysis of microRNA (miR) expression in the central nervous system white matter of SJL mice infected with the BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) revealed a significant reduction of miR-219, a critical regulator of myelin assembly and repair. Restoration of miR-219 expression by intranasal administration of a synthetic miR-219 mimic before disease onset ameliorates clinical disease, reduces neurogliosis, and partially recovers motor and sensorimotor function by negatively regulating proinflammatory cytokines and virus RNA replication. Moreover, RNA sequencing of host lesions showed that miR-219 significantly downregulated two genes essential for the biosynthetic cholesterol pathway, Cyp51 (lanosterol 14-α-demethylase) and Srebf1 (sterol regulatory element-binding protein-1), and reduced cholesterol biosynthesis in infected mice and rat CG-4 glial precursor cells in culture. The change in cholesterol biosynthesis had both anti-inflammatory and anti-viral effects. Because RNA viruses hijack endoplasmic reticulum double-layered membranes to provide a platform for RNA virus replication and are dependent on endogenous pools of cholesterol, miR-219 interference with cholesterol biosynthesis interfered virus RNA replication. These findings demonstrate that miR-219 inhibits TMEV-induced demyelinating disease through its anti-inflammatory and anti-viral properties.

SJL bone marrow-derived macrophages do not have IRF3 mutations and are highly susceptible to Theiler's virus infection.

It is well known that SJL mice are susceptible to Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease while C57BL6 (B6) and B10 mice are resistant, and H-2s on a B10 background (B10.S) contributes modestly to susceptibility. A recent study linked two IRF3 non-conservative mutations in SJL compared to B10.S mice to resistance to TMEV infection of SJL peritoneal-derived macrophages, an observation of practical interest in light of the central role of IRF3 transcription factor in the type I interferon (IFN) response. However, we did not find these non-conservative mutations among SJL, B10.S, B6 and B10 mice in the IRF3 amino acid sequence, and show SJL bone marrow-derived macrophages infected with TMEV exhibit increased virus RNA replication and infectious virus yields as well as greater IL-6 production than C57Bl strain (including B10.S) cultures.

During Infection, Theiler's Virions Are Cleaved by Caspases and Disassembled into Pentamers.

UNLABELLED: Infected macrophages in spinal cords of mice persistently infected with Theiler's murine encephalomyelitis virus (TMEV) undergo apoptosis, resulting in restricted virus yields, as do infected macrophages in culture. Apoptosis of murine macrophages in culture occurs via the intrinsic pathway later in infection (>10 h postinfection [p.i.]) after maximal virus titers (150 to 200 PFU/cell) have been reached, with loss of most infectious virus (<5 PFU/cell) by 20 to 24 h p.i. Here, we show that BeAn virus RNA replication, translation, polyprotein processing into final protein products, and assembly of protomers and pentamers in infected M1-D macrophages did not differ from those processes in TMEV-infected BHK-21 cells, which undergo necroptosis. However, the initial difference from BHK-21 cell infection was seen at 10 to 12 h p.i., where virions from the 160S peak in sucrose gradients had incompletely processed VP0 (compared to that in infected BHK-21 cells). Thereafter, there was a gradual loss of the 160S virion peak in sucrose gradients, with replacement by a 216S peak that was observed to contain pentamers among lipid debris in negatively stained grids by electron microscopy. After infection or incubation of purified virions with activated caspase-3 in vitro, 13- and 17-kDa capsid peptide fragments were observed and were predicted by algorithms to contain cleavage sites within proteins by cysteine-dependent aspartate-directed proteases. These findings suggest that caspase cleavage of sites in exposed capsid loops of assembled virions occurs contemporaneously with the onset and progression of apoptosis later in the infection. IMPORTANCE: Theiler's murine encephalomyelitis virus (TMEV) infection in mice results in establishment of virus persistence in the central nervous system and chronic inflammatory demyelinating disease, providing an experimental animal model for multiple sclerosis. Virus persistence takes place primarily in macrophages recruited into the spinal cord that undergo apoptosis and in turn may facilitate viral spread via infected apoptotic blebs. Infection of murine macrophages in culture results in restricted virus yields late in infection. Here it is shown that the early steps of the virus life cycle in infected macrophages in vitro do not differ from these processes in TMEV-infected BHK-21 cells, which undergo necroptosis. However, the findings late in infection suggest that caspases cleave sites in exposed capsid loops and possibly internal sites of assembled virions occurring contemporaneously with onset and progression of apoptosis. Mechanistically, this would explain the dramatic loss in virus yields during TMEV-induced apoptosis and attenuate the virus, enabling persistence.

Double-Stranded RNA Is Detected by Immunofluorescence Analysis in RNA and DNA Virus Infections, Including Those by Negative-Stranded RNA Viruses.

UNLABELLED: Early biochemical studies of viral replication suggested that most viruses produce double-stranded RNA (dsRNA), which is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in double-stranded DNA and positive-strand RNA virus infections but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any, dsRNA or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in the use of dsRNA antibodies for virus discovery, particularly in pathological specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected the in situ formation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus, and Nyamanini virus, which represent viruses from different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice (MVM), a single-stranded DNA (ssDNA) parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm, it was also seen in the nucleus of cells infected with influenza A virus, Nyamanini virus, and MVM. Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. The apoptosis induced in several uninfected cell lines failed to upregulate dsRNA formation. IMPORTANCE: An effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is a viral product essential for the induction of innate immunity, leading to the production of type I interferons (IFNs) and the activation of hundreds of IFN-stimulated genes. The present study demonstrates that infections, including those by ssDNA viruses and positive- and negative-strand RNA viruses, produce dsRNAs detectable by standard immunofluorescence staining. While dsRNA staining was primarily observed in the cytoplasm, nuclear staining was also present in some RNA and DNA virus infections. The nucleus is unlikely to have pathogen-associated molecular pattern (PAMP) receptors for dsRNA because of the presence of host dsRNA molecules. Thus, it is likely that most animal virus infections produce dsRNA species detectable by immunofluorescence staining, which may prove useful in viral discovery as well.

Inhibition of Theiler's virus-induced apoptosis in infected murine macrophages results in necroptosis.

In mice Theiler's murine encephalomyelitis virus (TMEV) persists in macrophages that eventually undergo apoptosis. TMEV infection of macrophages in culture induces apoptosis through the intrinsic pathway, restricting virus yields. We show that inhibition of TMEV-induced apoptosis leads to phosphorylation of receptor interacting protein 1 (RIP1), localization of RIP1 and RIP3 to mitochondria, ROS production independent of MAPK activation and programmed necrosis (necroptosis). Blocking both apoptosis and necroptosis restored virus yields.

Mutation of the Theiler's virus leader protein zinc-finger domain impairs apoptotic activity in murine macrophages.

The Theiler's murine encephalomyelitis virus (TMEV) leader (L) protein zinc-finger domain was mutated to study its role in cell death in infection of the murine macrophage cell line M1-D, revealing that an intact zinc-finger domain is required for full apoptotic activity. A functional L zinc-finger domain was also required for activation of p38 MAPK that results in phosphorylation and activation of p53, and in turn, alteration of the conformation of the anti-apoptotic proteins Puma and Mcl-1, leading to the release of pro-apoptotic Bax and apoptosis through the intrinsic pathway. TMEV infection also inhibits host protein synthesis, a stress shown by others to induce apoptosis. Since inhibition of host protein synthesis follows rather than precedes activation of MKK3/6 and p38, it seems less likely that it triggers apoptosis in infected cells. Finally, we showed that the levels of reactive oxygen species following infection were consistent with apoptotic rather than necrotic cell death. Thus, these experiments support an important role for the TMEV L protein zinc-finger domain in apoptosis in an infected murine macrophage line.

The antiapoptotic protein Mcl-1 controls the type of cell death in Theiler's virus-infected BHK-21 cells.

Theiler's murine encephalomyelitis virus (TMEV) results in a persistent central nervous system infection (CNS) and immune-mediated demyelination in mice. TMEV largely persists in macrophages (Ms) in the CNS, and infected Ms in vitro undergo apoptosis, whereas the infection of other rodent cells produces necrosis. We have found that necrosis is the dominant form of cell death in BeAn virus-infected BHK-21 cells but that ~20% of cells undergo apoptosis. Mcl-1 was highly expressed in BHK-21 cells, and protein levels decreased upon infection, consistent with onset of apoptosis. In infected BHK-21 cells in which Mcl-1 expression was knocked down using silencing RNAs there was a 3-fold increase in apoptotic cell death compared to parental cells. The apoptotic program switched on by BeAn virus is similar to that in mouse Ms, with hallmarks of activation of the intrinsic apoptotic pathway in a tumor suppressor protein p53-dependent manner. Infection of stable Mcl-1-knockdown cells led to restricted virus titers and increased physical to infectious particle (PFU) ratios, with additional data suggesting that a late step in the viral life cycle after viral RNA replication, protein synthesis, and polyprotein processing is affected by apoptosis. Together, these results indicate that Mcl-1 acts as a critical prosurvival factor that protects against apoptosis and allows high yields of infectious virus in BHK-21 cells.

Activation of tumor suppressor protein p53 is required for Theiler's murine encephalomyelitis virus-induced apoptosis in M1-D macrophages.

Theiler's murine encephalomyelitis virus (TMEV) is a highly cytolytic picornavirus that persists in the mouse central nervous system (CNS) largely in macrophages with infection maintained by macrophage-to-macrophage spread. Infected macrophages in the CNS undergo apoptosis. We recently showed that M1-D macrophages infected with the low-neurovirulence TMEV BeAn virus became apoptotic through the mitochondrial pathway that is Bax mediated. Our present analyses of the molecular events and signaling pathway(s) culminating in the mitochondrial outer membrane permeabilization that initiates the caspase cascade and apoptosis of BeAn virus-infected M1-D macrophages revealed activation of p38 mitogen-activated protein kinase by 2 to 3 h postinfection (p.i.), followed by phosphorylation of tumor suppressor protein p53 Ser 15 at 3 to 6 h p.i., stabilizing p53 levels until 6 h p.i. Activated p53 upregulated the transcription of proapoptotic puma and noxa genes at 2 to 4 h p.i. and their BH3-only protein expression, followed by the loss of detectable prosurvival Mcl-1 and A1 proteins at 4 to 10 h p.i. Degradation of the prosurvival proteins is known to release Bax, which forms homo-oligomers and translocates into and permeabilizes the mitochondrial outer membrane. Inhibition of phospho-p38 by two specific inhibitors, SB203580 and BIRB796, led to a significant decrease in apoptosis at 10 h p.i., with no effect on virus titers (only SB203580 tested). Together, these data indicate that p53 activation is required for the induction of apoptosis in infected M1-D cells.

Theiler's murine encephalomyelitis virus leader protein is the only nonstructural protein tested that induces apoptosis when transfected into mammalian cells.

Theiler's murine encephalomyelitis virus (TMEV) induces two distinct cell death programs, necrosis and apoptosis. The apoptotic pathway is of particular interest because TMEV persists in the central nervous system of mice, largely in infiltrating macrophages, which undergo apoptosis. Infection of murine macrophages in culture induces apoptosis that is Bax dependent through the intrinsic or mitochondrial pathway, restricting infectious-virus yields and raising the possibility that apoptosis represents a mechanism to attenuate TMEV yet promote macrophage-to-macrophage spread during persistent infection. To help define the cellular stressors and upstream signaling events leading to apoptosis during TMEV infection, we screened baby hamster kidney (BHK-21) cells transfected to express individual nonstructural genes (except 3B) of the low-neurovirulence BeAn virus strain for cell death. Only expression of the leader protein led to apoptosis, as assessed by fluorescence-activated cell sorting analysis of propidium iodide- and annexin V-stained transfected cells, immunoblot analysis of poly(ADP-ribose) polymerase and caspase cleavages, electron microscopy, and inhibition of apoptosis by the pancaspase inhibitor qVD-OPh. After transfection, Bak and not Bax expression increased, suggesting that the apical pathway leading to activation of these Bcl-2 multi-BH-domain proapoptotic proteins differs in BeAn virus infection versus L transfection. Mutation to remove the CHCC Zn finger motif from L, a motif required by L to mediate inhibition of nucleocytoplasmic trafficking, significantly reduced L-protein-induced apoptosis in both BHK-21 and M1-D macrophages.

Theiler's virus-induced intrinsic apoptosis in M1-D macrophages is Bax mediated and restricts virus infectivity: a mechanism for persistence of a cytolytic virus.

Theiler's murine encephalomyelitis virus (TMEV), a member of the Cardiovirus genus in the family Picornaviridae, is a highly cytolytic virus that produces necrotic death in rodent cells except for macrophages, which undergo apoptosis. In the present study we have analyzed the kinetics of BeAn virus infection in M1-D cells, in order to temporally relate virus replication to the apoptotic signaling events. Apoptosis was associated with early exponential virus growth from 1 to 12 h postinfection (p.i.); however, >/=80% of peak infectivity was lost by 16 to 24 h p.i. The pan-caspase inhibitor qVD-OPh led to significantly higher virus yields, while zVAD-fmk completely inhibited virus replication until 10 h p.i., precluding its assessment in apoptosis. In contrast, while zVAD-fmk significantly inhibited BeAn virus replication in BHK-21 cells at 12 and 16 h p.i., virus replication at these time points was not altered by qVD-OPh. Bax translocation into mitochondria, efflux of cytochrome c into the cytoplasm, and activation of caspases 9 and 3 between approximately 8 and 12 h p.i. (all hallmarks of the intrinsic apoptotic pathway) were transiently inhibited by expression of Bcl-2, which is not expressed in M1-D cells. Thus, BeAn virus infection in M1-D macrophages, which restricts virus replication, provides a potential mechanism for modulating TMEV neurovirulence during persistence in the mouse central nervous system.

A specific viral cause of multiple sclerosis: one virus, one disease.

"Multiple sclerosis is an autoimmune disease," is heard so often that it is widely accepted as fact by the current generation of students and physicians. Yet, although it is undisputed that multiple sclerosis (MS) is immune mediated, an autoimmune mechanism remains unproven. Immune-mediated tissue damage can also result from viral infections in which the host immune response is directed to viral rather than self proteins, or as a consequence of nonspecific or bystander immune responses that change the local cytokine environment. Increasing evidence suggests that poorly controlled host immune responses account for much of the tissue damage in chronic infections, and it has been postulated that a similar mechanism may underlie many chronic diseases with features suggestive of an infectious causative factor, including MS. A recent study suggesting that oligodendrocyte death accompanied by microglial activation is the primary event in new MS lesion formation, rather than lymphocyte infiltration, could change the current mindset almost exclusively focused on autoimmunity. This review presents the rationale for considering MS a single disease caused by one virus, as well as the anticipated pattern of a persistent central nervous system infection, the application of Koch's postulates to viral discovery in MS as the causative agent, and tissue culture-independent genotypic approaches to viral discovery in MS.

Promoter analysis of human CC chemokine CCL23 gene in U937 monocytoid cells.

Expression of CCL23 is induced by external stimuli including PMA in monocytes, but its transcriptional regulation has not been studied to date. Serial deletion analysis of its 5' flanking region revealed that the region -293 to +31 was important for induction by PMA. Cis-acting elements at the -269/-264 (NFAT site), -167/-159 (NF-kappaB site), and -51/-43 (AP-1 site) positions were identified as the critical sites for the CCL23 expression in U937 cells. We demonstrated the binding of the transcription factors to the consensus sites. Specific inhibitors for signal pathways reduced PMA-induced expression of CCL23, confirming involvement of these transcription factors.

Human lactoferrin upregulates expression of KDR/Flk-1 and stimulates VEGF-A-mediated endothelial cell proliferation and migration.

Lactoferrin (LF) is a multifunctional iron-binding glycoprotein, which plays a variety of biological processes including immunity. In this study, we demonstrate that human LF upregulates KDR/Flk-1 mRNA and protein levels in HUVECs at an optimal concentration of 5 microg/ml, which subsequently promotes the VEGF-induced proliferation and migration of the endothelial cells. Exposure of HUVECs to LF significantly increased VEGF-induced ERK MAP kinase phosphorylation. The maximal stimulation of KDR/Flk-1 expression by LF was correlated with LF-induced increase in cell proliferation and migration. These findings suggest that LF may stimulate in vivo angiogenesis via upregulation of KDR/Flk-1 expression in endothelial cells.