Sally E. Stringer

Axon Sorting in the Optic Tract Requires HSPG Synthesis by ext2 (dackel) and extl3 (boxer)

Retinal ganglion cell (RGC) axons are topographically ordered in the optic tract according to their retinal origin. In zebrafish dackel (dak) and boxer (box) mutants, some dorsal RGC axons missort in the optic tract but innervate the tectum topographically. Molecular cloning reveals that dak and box encode ext2 and extl3, glycosyltransferases implicated in heparan sulfate (HS) biosynthesis. Both genes are required for HS synthesis, as shown by biochemical and immunohistochemical analysis, and are expressed maternally and then ubiquitously, likely playing permissive roles. Missorting in box can be rescued by overexpression of extl3. dak;box double mutants show synthetic pathfinding phenotypes that phenocopy robo2 mutants, suggesting that Robo2 function requires HS in vivo; however, tract sorting does not require Robo function, since it is normal in robo2 null mutants. This genetic evidence that heparan sulfate proteoglycan function is required for optic tract sorting provides clues to begin understanding the underlying molecular mechanisms.

Endothelial progenitor cells enter the aging arena

Age is a significant risk factor for the development of vascular diseases, such as atherosclerosis. Although pharmacological treatments, including statins and anti-hypertensive drugs, have improved the prognosis for patients with cardiovascular disease, it remains a leading cause of mortality in those aged 65 years and over. Furthermore, given the increased life expectancy of the population in developed countries, there is a clear need for alternative treatment strategies. Consequently, the relationship between aging and progenitor cell-mediated repair is of great interest. Endothelial progenitor cells (EPCs) play an integral role in the cellular repair mechanisms for endothelial regeneration and maintenance. However, EPCs are subject to age-associated changes that diminish their number in circulation and function, thereby enhancing vascular disease risk. A great deal of research is aimed at developing strategies to harness the regenerative capacity of these cells. In this review, we discuss the current understanding of the cells termed “EPCs,” examine the impact of age on EPC-mediated repair and identify therapeutic targets with potential for attenuating the age-related decline in vascular health via beneficial actions on EPCs.

Activation of Pak1/Akt/eNOS signaling following sphingosine-1-phosphate release as part of a mechanism protecting cardiomyocytes against ischemic cell injury.

We investigated whether plasma long-chain sphingoid base (LCSB) concentrations are altered by transient cardiac ischemia during percutaneous coronary intervention (PCI) in humans and examined the signaling through the sphingosine-1-phosphate (S1P) cascade as a mechanism underlying the S1P cardioprotective effect in cardiac myocytes. Venous samples were collected from either the coronary sinus (n = 7) or femoral vein (n = 24) of 31 patients at 1 and 5 min and 12 h, following induction of transient myocardial ischemia during elective PCI. Coronary sinus levels of LCSB were increased by 1,072% at 1 min and 941% at 5 min (n = 7), while peripheral blood levels of LCSB were increased by 579% at 1 min, 617% at 5 min, and 436% at 12 h (n = 24). In cultured cardiac myocytes, S1P, sphingosine (SPH), and FTY720, a sphingolipid drug candidate, showed protective effects against CoCl induced hypoxia/ischemic cell injury by reducing lactate dehydrogenase activity. Twenty-five nanomolars of FTY720 significantly increased phospho-Pak1 and phospho-Akt levels by 56 and 65.6% in cells treated with this drug for 15 min. Further experiments demonstrated that FTY720 triggered nitric oxide release from cardiac myocytes is through pertussis toxin-sensitive phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase signaling. In ex vivo hearts, ischemic preconditioning was cardioprotective in wild-type control mice (Pak1f/f), but this protection appeared to be ineffective in cardiomyocyte-specific Pak1 knockout (Pak1cko) hearts. The present study provides the first direct evidence of the behavior of plasma sphingolipids following transient cardiac ischemia with dramatic and early increases in LCSB in humans. We also demonstrated that S1P, SPH, and FTY720 have protective effects against hypoxic/ischemic cell injury, likely a Pak1/Akt1 signaling cascade and nitric oxide release. Further study on a mouse model of cardiac specific deletion of Pak1 demonstrates a crucial role of Pak1 in cardiac protection against ischemia/reperfusion injury.

Decorin GAG Synthesis and TGF-β Signaling Mediate Ox-LDL–Induced Mineralization of Human Vascular Smooth Muscle Cells

Objective—Decorin and oxidized low-density lipoprotein (Ox-LDL) independently induce osteogenic differentiation of vascular smooth muscle cells (VSMCs). We aimed to determine whether decorin glycosaminoglycan (GAG) chain synthesis contributes to Ox-LDL–induced differentiation and calcification of human VSMCs in vitro. Methods and Results—Human VSMCs treated with Ox-LDL to induce oxidative stress showed increased alkaline phosphatase (ALP) activity, accelerated mineralization, and a difference in both decorin GAG chain biosynthesis and CS/DS structure compared with untreated controls. Ox-LDL increased mRNA abundance of both xylosyltransferase (XT)-I, the key enzyme responsible for GAG chain biosynthesis and Msx2, a marker of osteogenic differentiation. Furthermore, downregulation of XT-I expression using small interfering RNA blocked Ox-LDL–induced VSMC mineralization. Adenoviral-mediated overexpression of decorin, but not a mutated unglycanated form, accelerated mineralization of VSMCs, suggesting GAG chain addition on decorin is crucial for the process of differentiation. The decorin-induced VSMC osteogenic differentiation involved activation of the transforming growth factor (TGF)-&bgr; pathway, because it was attenuated by blocking of TGF-&bgr; receptor signaling and because decorin overexpression potentiated phosphorylation of the downstream signaling molecule smad2. Conclusion—These studies provide direct evidence that oxidative stress–mediated decorin GAG chain synthesis triggers TGF-&bgr; signaling and mineralization of VSMCs in vitro.

No haploinsufficiency but loss of heterozygosity for EXT in multiple osteochondromas.

Multiple osteochondromas (MO) is an autosomal dominant disorder caused by germline mutations in EXT1 and/or EXT2. In contrast, solitary osteochondroma (SO) is nonhereditary. Products of the EXT gene are involved in heparan sulfate (HS) biosynthesis. In this study, we investigated whether osteochondromas arise via either loss of heterozygosity (2 hits) or haploinsufficiency. An in vitro three-dimensional chondrogenic pellet model was used to compare heterozygous bone marrow-derived mesenchymal stem cells (MSCs EXT(wt/-)) of MO patients with normal MSCs and the corresponding tumor specimens (presumed EXT(-/-)). We demonstrated a second hit in EXT in five of eight osteochondromas. HS chain length and structure, in vitro chondrogenesis, and EXT expression levels were identical in both EXT(wt/-) and normal MSCs. Immunohistochemistry for HS, HS proteoglycans, and HS-dependent signaling pathways (eg, TGF-β/BMP, Wnt, and PTHLH) also showed no differences. The cartilaginous cap of osteochondroma contained a mixture of HS-positive and HS-negative cells. Because a heterozygous EXT mutation does not affect chondrogenesis, EXT, HS, or downstream signaling pathways in MSCs, our results refute the haploinsufficiency theory. We found a second hit in 63% of analyzed osteochondromas, supporting the hypothesis that osteochondromas arise via loss of heterozygosity. The detection of the second hit may depend on the ratio of HS-positive (normal) versus HS-negative (mutated) cells in the cartilaginous cap of the osteochondroma.

Age-related impairment of endothelial progenitor cell migration correlates with structural alterations of heparan sulfate proteoglycans.

Aging poses one of the largest risk factors for the development of cardiovascular disease. The increased propensity toward vascular pathology with advancing age maybe explained, in part, by a reduction in the ability of circulating endothelial progenitor cells to contribute to vascular repair and regeneration. Although there is evidence to suggest that colony forming unit‐Hill cells and circulating angiogenic cells are subject to age‐associated changes that impair their function, the impact of aging on human outgrowth endothelial cell (OEC) function has been less studied. We demonstrate that OECs isolated from cord blood or peripheral blood samples from young and old individuals exhibit different characteristics in terms of their migratory capacity. In addition, age‐related structural changes were discovered in OEC heparan sulfate (HS), a glycocalyx component that is essential in many signalling pathways. An age‐associated decline in the migratory response of OECs toward a gradient of VEGF significantly correlated with a reduction in the relative percentage of the trisulfated disaccharide, 2‐O‐sulfated‐uronic acid, N, 6‐O‐sulfated‐glucosamine (UA[2S]‐GlcNS[6S]), within OEC cell surface HS polysaccharide chains. Furthermore, disruption of cell surface HS reduced the migratory response of peripheral blood‐derived OECs isolated from young subjects to levels similar to that observed for OECs from older individuals. Together these findings suggest that aging is associated with alterations in the fine structure of HS on the cell surface of OECs. Such changes may modulate the migration, homing, and engraftment capacity of these repair cells, thereby contributing to the progression of endothelial dysfunction and age‐related vascular pathologies.

Serum sphingolipids level as a novel potential marker for early detection of human myocardial ischaemic injury.

Background: Ventricular tachyarrhythmias are the most common and often the first manifestation of coronary heart disease and lead to sudden cardiac death (SCD). Early detection/identification of acute myocardial ischaemic injury at risk for malignant ventricular arrhythmias in patients remains an unmet medical need. In the present study, we examined the sphingolipids level after transient cardiac ischaemia following temporary coronary artery occlusion during percutaneous coronary intervention (PCI) in patients and determined the role of sphingolipids level as a novel marker for early detection of human myocardial ischaemic injury. Methods and Results: Venous samples were collected from either the coronary sinus (n = 7) or femoral vein (n = 24) from 31 patients aged 40–73 years-old at 1, 5 min, and 12 h, following elective PCI. Plasma sphingolipids levels were assessed by HPLC. At 1 min coronary sinus levels of sphingosine 1-phosphate (S1P), sphingosine (SPH), and sphinganine (SA) were increased by 314, 115, and 614%, respectively (n = 7), while peripheral blood levels increased by 79, 68, and 272% (n = 24). By 5 min, coronary sinus S1P and SPH levels increased further (720%, 117%), as did peripheral levels of S1P alone (792%). Where troponin T was detectable at 12 h (10 of 31), a strong correlation was found with peak S1P (R2 = 0.818; P < 0.0001). Conclusion: For the first time, we demonstrate the behavior of plasma sphingolipids following transient cardiac ischaemia in humans. The observation supports the important role of sphingolipids level as a potential novel marker of transient or prolonged myocardial ischaemia.

Cartilage tumour progression is characterized by an increased expression of heparan sulphate 6O-sulphation-modifying enzymes

Chondrosarcomas are malignant cartilage-forming tumours that can arise centrally (in the medulla) or peripherally (at the surface) of the bone. They are classified into three histological grades which correspond to the clinical severity. Previous studies by our group have shown altered signal transduction of the fibroblast growth factor and Wnt signalling pathways during peripheral chondrosarcoma progression. Heparan sulphate (HS) is a glycosaminoglycan that facilitates receptor binding of multiple growth factors, in which the sulphation of 6O position plays a pivotal role. 6O-Sulphation occurs through three HS 6O-sulphotransferases (HS6ST1-3) and is fine-tuned by two endosulphatases (SULF1-2) that remove 6O-sulphate groups. We have investigated whether the expression of HS6STs and SULFs changes during chondrosarcoma progression and have determined 6O-sulphation levels in two chondrosarcoma cell lines. Immunohistochemistry on tissue microarrays of chondrosarcomas showed that HS6ST3 and SULF1 were highly expressed in most chondrosarcomas, whereas SULF2 expression was absent in most cases. HS6ST1 and HS6ST2 expression are significantly increased during chondrosarcoma progression, which suggest that 6O-sulphation is increased during progression. This was confirmed in one grade III chondrosarcoma cell line, which showed a dramatically increased 6O-sulphation compared to an articular chondrocyte cell line by HPLC; another cell line showed an increased expression of one 6O-sulphated HS disaccharide. In conclusion, our results show increased HS6ST1 and HS6ST2 expression during chondrosarcoma progression and increased HS 6O-sulphation in vitro. As 6O-sulphation plays an important role in signal transduction, altered HS6ST expression might be associated with changes in signal transduction pathways in chondrosarcoma progression.

Dynamic expression patterns of 6-O endosulfatases during zebrafish development suggest a subfunctionalisation event for sulf2

The 6‐O‐endosulfatase enzymes (Sulfs) edit the final sulfation pattern and function of heparan sulfate (HS) by removal of 6‐O‐sulfate groups from the chain. To date, two mammalian sulf genes have been identified that regulate many signalling pathways during embryonic development. In zebrafish a sulf1 ortholog and duplicate copies of the mammalian sulf2 gene, sulf2a and sulf2, have been identified, which contain conserved motifs characteristic of vertebrate sulf genes. Zebrafish sulf1 and sulf2a are broadly expressed in the central nervous system (CNS) and non‐neuronal tissue including heart, somite boundaries, olfactory system, and otic vesicle, whereas sulf2 expression is almost entirely restricted to the CNS. The duplicate copies of sulf2 have distinct expression patterns, which together mirror that of mouse sulf2, suggesting duplication in the teleost lineage has been followed by subfunctionalisation, whereby both genes need to be preserved by selection to ensure the ancestral genes expression profile and function is maintained. Developmental Dynamics 239:3312–3323, 2010.

40 Age-related modification of heparan sulphate proteoglycans on human endothelial progenitor cells

Accumulating evidence indicates that vascular repair by endothelial progenitor cells (EPCs) is impaired with age. However, the molecular mechanisms underlying this functional impairment are not understood. Cell-surface heparan sulphate (HS) proteoglycans, by virtue of specific sulphated domains within the glycosaminoglycan chain, are able to bind a variety of ligands essential for EPC mobilisation, homing and differentiation. We hypothesise that structural changes of HS on EPCs contribute to vascular dysfunction in age and disease. Using umbilical cord blood, and adult peripheral blood from young and old subjects, we routinely isolate a rare population of EPCs, termed outgrowth endothelial cells (OECs). These cells are highly proliferative, express a panel of endothelial but not haematopoietic markers, can ingest Ac-LDL and form tubes in Matrigel. Structural analysis of HS by high performance liquid chromatography (HPLC) demonstrates a reduction of 6-O-sulphation of HS on the surface of OECs with vascular age. There is 21.9% 6-O-sulphation of HS chains on cord blood OECs compared to 14.6% and 10.1% on adult peripheral blood OECs from young (20–30 years) and older (>55 years) subjects respectively. Moreover, these HS structural changes correlate with a decrease in the proliferative and migratory capacities of these cells. The effects of these changes on the response of EPCs to signalling molecules implicated in EPC mobilisation and homing (VEGF and SDF-1) are currently being investigated. Whether the impairment in function can be rescued by the addition of soluble heparin is also being assessed. This work could have clinical relevance for therapeutic angiogenesis in patients with limb ischemia or vascular damage.