Lianchun Wang

A myocardial lineage derives from Tbx18 epicardial cells

Understanding the origins and roles of cardiac progenitor cells is important for elucidating the pathogenesis of congenital and acquired heart diseases. Moreover, manipulation of cardiac myocyte progenitors has potential for cell-based repair strategies for various myocardial disorders. Here we report the identification in mouse of a previously unknown cardiac myocyte lineage that derives from the proepicardial organ. These progenitor cells, which express the T-box transcription factor Tbx18, migrate onto the outer cardiac surface to form the epicardium, and then make a substantial contribution to myocytes in the ventricular septum and the atrial and ventricular walls. Tbx18-expressing cardiac progenitors also give rise to cardiac fibroblasts and coronary smooth muscle cells. The pluripotency of Tbx18 proepicardial cells provides a theoretical framework for applying these progenitors to effect cardiac repair and regeneration.

Endothelial heparan sulfate deficiency impairs L-selectin- and chemokine-mediated neutrophil trafficking during inflammatory responses.

Here we have studied the involvement of endothelial heparan sulfate in inflammation by inactivating the enzyme N-acetyl glucosamine N-deacetylase–N-sulfotransferase-1 in endothelial cells and leukocytes, which is required for the addition of sulfate to the heparin sulfate chains. Mutant mice developed normally but showed impaired neutrophil infiltration in various inflammation models. These effects were due to changes in heparan sulfate specifically in endothelial cells. Decreased neutrophil infiltration was partially due to altered rolling velocity correlated with weaker binding of L-selectin to endothelial cells. Chemokine transcytosis across endothelial cells and presentation on the cell surface were also reduced, resulting in decreased neutrophil firm adhesion and migration. Thus, endothelial heparan sulfate has three functions in inflammation: by acting as a ligand for L-selectin during neutrophil rolling; in chemokine transcytosis; and by binding and presenting chemokines at the lumenal surface of the endothelium.

Adult mouse epicardium modulates myocardial injury by secreting paracrine factors

The epicardium makes essential cellular and paracrine contributions to the growth of the fetal myocardium and the formation of the coronary vasculature. However, whether the epicardium has similar roles postnatally in the normal and injured heart remains enigmatic. Here, we have investigated this question using genetic fate-mapping approaches in mice. In uninjured postnatal heart, epicardial cells were quiescent. Myocardial infarction increased epicardial cell proliferation and stimulated formation of epicardium-derived cells (EPDCs), which remained in a thickened layer on the surface of the heart. EPDCs did not adopt cardiomyocyte or coronary EC fates, but rather differentiated into mesenchymal cells expressing fibroblast and smooth muscle cell markers. In vitro and in vivo assays demonstrated that EPDCs secreted paracrine factors that strongly promoted angiogenesis. In a myocardial infarction model, EPDC-conditioned medium reduced infarct size and improved heart function. Our findings indicate that epicardium modulates the cardiac injury response by conditioning the subepicardial environment, potentially offering a new therapeutic strategy for cardiac protection.

Heparin’s anti-inflammatory effects require glucosamine 6- O -sulfation and are mediated by blockade of L- and P-selectins

Heparin has been used clinically as an anticoagulant and antithrombotic agent for over 60 years. Here we show that the potent anti-inflammatory property of heparin results primarily from blockade of P-selectin and L-selectin. Unfractionated heparin and chemically modified analogs were tested as inhibitors of selectin binding to immobilized sialyl Lewis(X) and of cell adhesion to immobilized selectins or thrombin-activated endothelial cells. Compared with unfractionated heparin, the modified heparinoids had inhibitory activity in this general order: over-O-sulfated heparin > heparin > 2-O,3-O-desulfated > or = N-desulfated/N-acetylated heparin > or = carboxyl-reduced heparin > or= N-,2-O,3-O-desulfated heparin >> 6-O-desulfated heparin. The heparinoids also showed similar differences in their ability to inhibit thioglycollate-induced peritonitis and oxazolone-induced delayed-type hypersensitivity. Mice deficient in P- or L-selectins showed impaired inflammation, which could be further reduced by heparin. However, heparin had no additional effect in mice deficient in both P- and L-selectins. We conclude that (a) heparins anti-inflammatory effects are mainly mediated by blocking P- and L-selectin-initiated cell adhesion; (b) the sulfate groups at C6 on the glucosamine residues play a critical role in selectin inhibition; and (c) some non-anticoagulant forms of heparin retain anti-inflammatory activity. Such analogs may prove useful as therapeutically effective inhibitors of inflammation.

Liver heparan sulfate proteoglycans mediate clearance of triglyceride-rich lipoproteins independently of LDL receptor family members

We examined the role of hepatic heparan sulfate in triglyceride-rich lipoprotein metabolism by inactivating the biosynthetic gene GlcNAc N-deacetylase/N-sulfotransferase 1 (Ndst1) in hepatocytes using the Cre-loxP system, which resulted in an approximately 50% reduction in sulfation of liver heparan sulfate. Mice were viable and healthy, but they accumulated triglyceride-rich lipoprotein particles containing apoB-100, apoB-48, apoE, and apoCI-IV. Compounding the mutation with LDL receptor deficiency caused enhanced accumulation of both cholesterol- and triglyceride-rich particles compared with mice lacking only LDL receptors, suggesting that heparan sulfate participates in the clearance of cholesterol-rich lipoproteins as well. Mutant mice synthesized VLDL normally but showed reduced plasma clearance of human VLDL and a corresponding reduction in hepatic VLDL uptake. Retinyl ester excursion studies revealed that clearance of intestinally derived lipoproteins also depended on hepatocyte heparan sulfate. These findings show that under normal physiological conditions, hepatic heparan sulfate proteoglycans play a crucial role in the clearance of both intestinally derived and hepatic lipoprotein particles.

Evolutionary Differences in Glycosaminoglycan Fine Structure Detected by Quantitative Glycan Reductive Isotope Labeling

To facilitate qualitative and quantitative analysis of glycosaminoglycans, we tagged the reducing end of lyase-generated disaccharides with aniline-containing stable isotopes (12C6 and 13C6). Because different isotope tags have no effect on chromatographic retention times but can be discriminated by a mass detector, differentially isotope-tagged samples can be compared simultaneously by liquid chromatography/mass spectrometry and quantified by admixture with known amounts of standards. The technique is adaptable to all types of glycosaminoglycans, and its sensitivity is only limited by the type of mass spectrometer available. We validated the method using commercial heparin and keratan sulfate as well as heparan sulfate isolated from mutant and wild-type Chinese hamster ovary cells, and select tissues from mutant and wild-type mice. This new method provides more robust, reliable, and sensitive means of quantitative evaluation of glycosaminoglycan disaccharide compositions than existing techniques allowing us to compare the chondroitin and heparan sulfate compositions of Hydra vulgaris, Drosophila melanogaster, Caenorhabditis elegans, and mammalian cells. Our results demonstrate significant differences in glycosaminoglycan structure among these organisms that might represent evolutionarily distinct functional motifs.

Selectin Blocking Activity of a Fucosylated Chondroitin Sulfate Glycosaminoglycan from Sea Cucumber EFFECT ON TUMOR METASTASIS AND NEUTROPHIL RECRUITMENT

Heparin is an excellent inhibitor of P- and L-selectin binding to the carbohydrate determinant, sialyl Lewisx. As a consequence of its anti-selectin activity, heparin attenuates metastasis and inflammation. Here we show that fucosylated chondroitin sulfate (FucCS), a polysaccharide isolated from sea cucumber composed of a chondroitin sulfate backbone substituted at the 3-position of the β-d-glucuronic acid residues with 2,4-disulfated α-l-fucopyranosyl branches, is a potent inhibitor of P- and L-selectin binding to immobilized sialyl Lewisx and LS180 carcinoma cell attachment to immobilized P- and L-selectins. Inhibition occurs in a concentration-dependent manner. Furthermore, FucCS was 4–8-fold more potent than heparin in the inhibition of the P- and L-selectin-sialyl Lewisx interactions. No inhibition of E-selectin was observed. FucCS also inhibited lung colonization by adenocarcinoma MC-38 cells in an experimental metastasis model in mice, as well as neutrophil recruitment in two models of inflammation (thioglycollate-induced peritonitis and lipopolysaccharide-induced lung inflammation). Inhibition occurred at a dose that produces no significant change in plasma activated partial thromboplastin time. Removal of the sulfated fucose branches on the FucCS abolished the inhibitory effect in vitro and in vivo. Overall, the results suggest that invertebrate FucCS may be a potential alternative to heparin for blocking metastasis and inflammatory reactions without the undesirable side effects of anticoagulant heparin.

Surfen, a small molecule antagonist of heparan sulfate

In a search for small molecule antagonists of heparan sulfate, we examined the activity of bis-2-methyl-4-amino-quinolyl-6-carbamide, also known as surfen. Fluorescence-based titrations indicated that surfen bound to glycosaminoglycans, and the extent of binding increased according to charge density in the order heparin > dermatan sulfate > heparan sulfate > chondroitin sulfate. All charged groups in heparin (N-sulfates, O-sulfates, and carboxyl groups) contributed to binding, consistent with the idea that surfen interacted electrostatically. Surfen neutralized the anticoagulant activity of both unfractionated and low molecular weight heparins and inhibited enzymatic sulfation and degradation reactions in vitro. Addition of surfen to cultured cells blocked FGF2-binding and signaling that depended on cell surface heparan sulfate and prevented both FGF2- and VEGF165-mediated sprouting of endothelial cells in Matrigel. Surfen also blocked heparan sulfate-mediated cell adhesion to the Hep-II domain of fibronectin and prevented infection by HSV-1 that depended on glycoprotein D interaction with heparan sulfate. These findings demonstrate the feasibility of identifying small molecule antagonists of heparan sulfate and raise the possibility of developing pharmacological agents to treat disorders that involve glycosaminoglycan–protein interactions.

Contribution of Proteoglycans to Human Immunodeficiency Virus Type 1 Brain Invasion

ABSTRACT As a neurotropic virus, human immunodeficiency virus type 1 (HIV-1) invades the brain and causes severe neuronal, astrocyte, and myelin damage in AIDS patients. To gain access to the brain, HIV-1 must migrate through brain microvascular endothelial cells (BMECs), which compose the blood-brain barrier (BBB). Given that BMECs lack the entry receptor CD4, HIV-1 must use receptors distinct from CD4 to enter these cells. We previously reported that cell surface proteoglycans serve as major HIV-1 receptors on primary human endothelial cells. In this study, we examined whether proteoglycans also impact cell-free HIV-1 invasion of the brain. Using an artificial BBB transmigration assay, we found that both heparan and chondroitin sulfate proteoglycans (HSPGs and CSPGs, respectively) are abundantly expressed on primary BMECs and promote HIV-1 attachment and entry. In contrast, the classical entry receptors, CXCR4 and CCR5, only moderately enhanced these processes. HSPGs and CSPGs captured HIV-1 in a gp120-dependent manner. However, no correlation between coreceptor usage and transmigration was identified. Furthermore, brain-derived viruses did not transmigrate more efficiently than lymphoid-derived viruses, suggesting that the ability of HIV-1 to replicate in the brain does not correlate with its capacity to migrate through the BBB as cell-free virus. Given that HIV-1-proteoglycan interactions are based on electrostatic contacts between basic residues in gp120 and sulfate groups in proteoglycans, HIV-1 may exploit these interactions to rapidly enter and migrate through the BBB to invade the brain.

Tumor Vasculature Targeted Photodynamic Therapy for Enhanced Delivery of Nanoparticles

Delivery of nanoparticle drugs to tumors relies heavily on the enhanced permeability and retention (EPR) effect. While many consider the effect to be equally effective on all tumors, it varies drastically among the tumors’ origins, stages, and organs, owing much to differences in vessel leakiness. Suboptimal EPR effect represents a major problem in the translation of nanomedicine to the clinic. In the present study, we introduce a photodynamic therapy (PDT)-based EPR enhancement technology. The method uses RGD-modified ferritin (RFRT) as “smart” carriers that site-specifically deliver 1O2 to the tumor endothelium. The photodynamic stimulus can cause permeabilized tumor vessels that facilitate extravasation of nanoparticles at the sites. The method has proven to be safe, selective, and effective. Increased tumor uptake was observed with a wide range of nanoparticles by as much as 20.08-fold. It is expected that the methodology can find wide applications in the area of nanomedicine.