Low Molecular Weight Heparan Sulfate Research Articles

TREM2 on microglia cell surface binds to and forms functional binary complexes with heparan sulfate modified with 6-O-sulfation and iduronic acid

The Triggering Receptor Expressed on Myeloid Cells-2 (TREM2), a pivotal innate immune receptor, orchestrates functions such as inflammatory responses, phagocytosis, cell survival, and neuroprotection. TREM2 variants R47H and R62H have been associated with Alzheimer's disease, yet the underlying mechanisms remain elusive. Our previous research established that TREM2 binds to heparan sulfate (HS) and variants R47H and R62H exhibit reduced affinity for HS. Building upon this groundwork, our current study delves into the interplay between TREM2 and HS and its impact on microglial function. We confirm TREM2's binding to cell surface HS and demonstrate that TREM2 interacts with HS, forming HS-TREM2 binary complexes on microglia cell surfaces. Employing various biochemical techniques, including Surface Plasmon Resonance, low molecular weight HS microarray screening, and serial HS mutant cell surface binding assays, we demonstrate TREM2's robust affinity for HS, and the effective binding requires a minimum HS size of approximately 10 saccharide units. Notably, TREM2 selectively binds specific HS structures, with 6-O-sulfation and, to a lesser extent, the iduronic acid residue playing crucial roles. N-sulfation and 2-O-sulfation are dispensable for this interaction. Furthermore, we reveal that 6-O-sulfation is essential for HS-TREM2 ternary complex formation on the microglial cell surface, and HS and its 6-O-sulfation are necessary for TREM2-mediated ApoE3 uptake in microglia. By delineating the interaction between HS and TREM2 on the microglial cell surface and demonstrating its role in facilitating TREM2-mediated ApoE uptake by microglia, our findings provide valuable insights that can inform targeted interventions for modulating microglial functions in Alzheimer's disease.

Exploring the importance of inclusion criteria beyond the color of skin: Improvement in facial redness in skin of color subjects—An open-label, single center clinical study utilizing low molecular weight heparan sulfate

To the Editor: The awareness of skin of color (SOC) in dermatology is expanding; however, a lack of diversity in dermatologic clinical trials persists,1Elston D.M. Letter from the editor: diversity and inclusion in clinical trials link to JAAD-D-21-03191.J Am Acad Dermatol. 2022; 87: 981-982https://doi.org/10.1016/j.jaad.2022.05.041Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar especially considering the inclusion of African Americans/Blacks.2Okeke C.A.V. Bhayana M. Simmonds F.C. et al.Improving African American enrollment in hidradenitis suppurativa clinical trials: a clinical and research staff perspective.J Am Acad Dermatol. 2021; 85: AB47https://doi.org/10.1016/J.JAAD.2021.06.215Abstract Full Text Full Text PDF Google Scholar,3Ferguson J.E. Vetos D. Ho B.V. et al.Trends in diversity of participants in dermatology clinical trials over time: a systematic review.J Am Acad Dermatol. 2022; 86: 1403-1405https://doi.org/10.1016/J.JAAD.2021.05.052Abstract Full Text Full Text PDF Scopus (0) Google Scholar This study provides evidence of how SOC can be excluded and/or underestimated for severity of symptoms because redness can be overlooked by visual examination. This open-label, single center clinical study assessing SOC using clinical grading and biophysical testing reveals the positive results and benefits of a daily regimen containing a patented low molecular weight heparan sulfate—heparan sulfate analog (HSA). This work highlights how SOC subjects, especially those with darker skin tones, may be omitted from study analyses due to visual clinical grading limitations. Twelve subjects, Fitzpatrick skin types III to VI (African American/Black [75%], American Indian or Alaska Native [16.7%], and Asian [8.3%]), were included in the intent-to-treat population. Subjects used HSA and cleanser twice per day (AM/PM) and sunscreen SPF 52 tinted or SPF 49 untinted at least once per day (AM). Clinical evaluations were conducted at visit 1 (baseline), visit 2 (week 2), and visit 3 (week 4). Subjects participated in the following procedures at each time point: clinical grading of efficacy parameters,4Griffiths C.E. Wang T.S. Hamilton T.A. Voorhees J.J. Ellis C.N. A photonumeric scale for the assessment of cutaneous photodamage.Arch Dermatol. 1992; 128: 347-351https://doi.org/10.1001/archderm.1992.01680130061006Crossref PubMed Scopus (199) Google Scholar tolerability evaluations, Mexameter measurements, VISIA-CR imaging, and self-assessment questionnaires. Another component of the study was to collect skin swabs, at baseline and week 4, to analyze the inflammatory markers IL-1α, IL-1RA, and PGE2. Notably, none of the SOC subjects qualified for the swab study because of the criteria of having visible moderate to severe facial redness. Although there was a decrease in local cutaneous global erythema at 4 weeks compared to baseline evaluated by signs and symptoms of clinically graded facial redness (Supplementary Fig 1, available via Mendeley at https://doi.org/10.17632/6bw7rjn2db.1, upper panel), this was not as apparent as the decrease in facial redness captured by bioinstrumentation measurements (Supplementary Fig 1, available via Mendeley at https://doi.org/10.17632/6bw7rjn2db.1, middle panel). The HSA regimen resulted in true reduction in redness as assessed by the Mexameter measurements (2.2% decrease in melanin, 5.6% decrease in hemoglobin) (Supplementary Fig 1, available via Mendeley at https://doi.org/10.17632/6bw7rjn2db.1, middle panel) indicating an improvement in erythema/facial redness at 4 weeks compared to baseline that was not captured to the same extent by visible clinical grading. This resulted in exclusion of these individuals from the inflammatory marker assessment due to the lack of apparent moderate to severe facial erythema by visible clinical grading. The self-assessment questionnaire and tolerability evaluations (Supplementary Fig 1, available via Mendeley at https://doi.org/10.17632/6bw7rjn2db.1, lower panel) revealed the regimen did not cause cutaneous irritation and was highly favorable among subjects. The HSA regimen tested substantially improved redness in SOC (Fig 1 and Supplementary Fig 2, available via Mendeley at https://doi.org/10.17632/tjxzcpjycp.1). However, due to visible grading inclusion criteria, these subjects were excluded from any analysis to correlate the reduction in redness to reduced inflammation. This highlights the critical need for careful consideration and evaluation of the various cutaneous presentations in SOC to ensure that underlying redness and potential inflammation are not overlooked in this population. The use of biophysical testing and visible grading promotes the inclusion and retention of SOC in clinical studies. Such actions will prevent explicit and unconscious biases, ensuring medical grade skin care products are generalizable to all. ASB is a consultant for Senté, Inc. ES-O, VDC, and DO have participated in Advisory Boards for Senté, Inc. SH and CAVO have no conflicts of interest to declare. We kindly thank all study participants. Also, we would like to thank Tanya Rhodes, PhD for her critical review of the manuscript and feedback. The clinical study (SGS Stephens Study Number: C21-P009) was conducted by SGS Stephens, Inc, Phoenix Research Center (2421 West Peoria Ave, Suite 124 Phoenix, AZ 85029).

34503 Improvements in facial redness and inflammation: An open-label, single center clinical study including low molecular weight heparan sulfate (HSA) in patients with sensitive skin

Background: Chronic inflammation damages barrier structure and skin homeostasis. Over 50% of the population reports self-declared sensitive skin. Heparan sulfate is a naturally occurring glycosaminoglycan, which has essential roles in skin functions. Here, we present the results of an open-label, single center clinical study testing a regimen created by Senté, Inc, which uses a patented low molecular weight heparan sulfate known as heparin sulfate analog (HSA). Methods: 46 subjects, Fitzpatrick skin types I-VI, were included in the intent-to-treat population. Subjects used HSA and Cleanser twice per day (AM/PM) and Sunscreen SPF 52 Tinted or SPF 49 Untinted at least once per day (AM). Clinical evaluations were conducted at visit 1 (baseline), visit 2 (week 2), and visit 3 (week 4). Subjects participated in the following procedures at each time point: Clinical Grading of Efficacy Parameters (4),Tolerability Evaluations, Mexameter Measurements, VISIA-CR Imaging, and Self-Assessment Questionnaires. Additionally, at baseline and week 4, skin swabs were collected from a subset of subjects to analyze the inflammatory markers IL-1α, IL-1RA, and PGE2. Results/conclusion: Overall results indicate that the regimen was effective showing a statistically significant improvement in facial redness. Likewise, there was a decrease in inflammation as indicated by a statistically significant downregulation in the aforementioned inflammatory markers. The facial products were well tolerated and well perceived by the subjects. This regimen addresses the unmet needs of those suffering from irritated and sensitive skin supported by clinical and scientific evidence.

Low molecular weight heparan sulfate containing facial skin care for reducing inflammation and restoring aged-skin homeostasis.

Chronic exposure of the skin to environmental factors, including solar radiation and air pollution, promotes skin aging by inducing inflammation which damages the skin. Heparan sulfate (HS) is one of the glycosaminoglycans, which has a central role in modulating skin repair. In its appearance in the skin, HS is large and highly polar and therefore unable to penetrate the skin. As a component in a topical formulation, the modified to a low molecular weight heparan sulfate analog (LMW-HS) showed biological modulation activity. This review discusses the potential role of topical formulations containing LMW-HS in restoring aged-skin homeostasis and skin health. An expert panel of dermatologists who regularly treat clinical signs of facial photoaging explored the role of LMW-HS containing formulations for reducing inflammation and facial-aging signs. For this purpose, evidence from the conducted literature searches was used together with expert opinion and experience of the panel. Extrinsic factors contribute to skin aging through oxidative stress, stimulating inflammation involved in extracellular matrix degradation. Evidence showed that chemokines require heparan sulfate for their full range of functional activities during innate immunity. Studies showed the LMW-HS containing topical formulation to penetrate the skin within 48hours of once-daily application. LMW-HS used in the periorbital area improved discoloration and wrinkles at Week 2 with continuous improvement up to Week 12. The LMW-HS containing formulation showed improvements in skin condition when applied on photo-damaged skin, indicating its therapeutic potential.

Global periorbital skin rejuvenation by a topical eye cream containing low molecular weight heparan sulfate (LMW-HS) and a blend of naturally derived extracts

Global periorbital skin rejuvenation by a topical eye cream containing low molecular weight heparan sulfate (LMW-HS) and a blend of naturally derived extracts

Concise chemoenzymatic synthesis of heparan sulfate analogues as potent BACE-1 inhibitors

Heparan sulfate (HS) and heparin, representative members of the glycosaminoglycans, possess distinct biological functions in terms of their specific interactions with hundreds of binding proteins. However, the structural properties of HS and heparin are complex due to their variable repeating motifs, different chain lengths and sulfation patterns. A concise chemoenzymatic approach has been developed to obtain well-defined low molecular weight (LMW) HS analogues. Pasteurella multocida heparosan synthase-2 (PmHS2) was utilized to fabricate the HS backbones with controllable chain lengths ranging from 14mer to 26mer. Moreover, regioselective and overall sulfation were conducted by chemical approach. The persulfated HS analogues exhibited more potent beta-site amyloid precursor protein (APP)-cleaving enzyme-1 (BACE-1) inhibitory activity than heparin and enoxaparin, and enhanced BACE-1 inhibitions were also found with the increasing molecular size of the HS analogues. This approach supplies the promising LMW HS analogues for the potential development of novel anti-Alzheimer's drugs.

Mental retardation in mucopolysaccharidoses correlates with high molecular weight urinary heparan sulphate derived glucosamine.

Mucopolysaccharidoses (MPS) are characterized by mental retardation constantly present in the severe forms of Hurler (MPS I), Hunter (MPS II) and Sanfilippo (MPS III) diseases. On the contrary, mental retardation is absent in Morquio (MPS IV) and Maroteaux-Lamy (MPS VI) diseases and absent or only minimal in the attenuated forms of MPS I, II and III. Considering that MPS patients affected by mental disease accumulate heparan sulfate (HS) due to specific enzymatic defects, we hypothesized a possible correlation between urinary HS-derived glucosamine (GlcN) accumulated in tissues and excreted in biological fluids and mental retardation. 83 healthy subjects were found to excrete HS in the form of fragments due to the activity of catabolic enzymes that are absent or impaired in MPS patients. On the contrary, urinary HS in 44 patients was observed to be composed of high molecular weight polymer and fragments of various lengths depending on MPS types. On this basis we correlated mental retardation with GlcN belonging to high and low molecular weight HS. We demonstrate a positive relationship between the accumulation of high molecular weight HS and mental retardation in MPS severe compared to attenuated forms. This is also supported by the consideration that accumulation of other GAGs different from HS, as in MPS IV and MPS VI, and low molecular weight HS fragments do not impact on central nervous system disease.

Identification of an autoantigen on the surface of apoptotic human T cells as a new protein interacting with inflammatory group IIA phospholipase A2

AbstractOne of the most studied secreted phospholipases A2 (sPLA2), the group IIA sPLA2, is found at high levels in inflammatory fluids of patients with autoimmune diseases. A characteristic of group IIA sPLA2 is its preference for negatively charged phospholipids, which become exposed on the extracellular leaflet of apoptotic cell membranes. We recently showed that low molecular weight heparan sulfate proteoglycans (HSPGs) and uncharacterized detergent-insoluble binding site(s) contribute to the enhanced binding of human group IIA PLA2 (hGIIA) to apoptotic human T cells. Using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry we now identify vimentin as the major HSPG-independent binding protein of hGIIA on apoptotic primary T lymphocytes. Vimentin is partially exposed on the surface of apoptotic T cells and binds hGIIA via its rod domain in a calcium-independent manner. Studies with hGIIA mutants showed that specific motifs in the interfacial binding surface are involved in the interaction with vimentin. The sPLA2 inhibitor LY311727, but not heparin, inhibited this interaction. In contrast, heparin but not LY311727 abrogated the binding of hGIIA to cellular HSPGs. Importantly, vimentin does not inhibit the catalytic activity of hGIIA. Altogether, the results show that vimentin, in conjunction with HSPGs, contributes to the enhanced binding of hGIIA to apoptotic T cells.

Do Heparins Do More than Just Treat Thrombosis? The Influence of Heparins on Cancer Spread

IntroductionThe influence of unfractionated heparin (UFH) and other anticoagulants on the spread of cancer has been reported since the early 1960s.1 However, clinical studies investigating the use of heparins in cancer patients have not produced consistent results.2 Intravenous, adjusted-dose UFH for 5 to 10 days has been the standard initial treatment for venous thrombosis. More recently, subcutaneous, fixed-dose, low molecular weight heparins (LMWHs), which are fractions of the parent compound, have been shown to be safe and effective alternatives to UFH in the initial treatment for venous thromboembolism.3-5 In one of our randomized clinical trials comparing LMWH and UFH in the initial treatment of deep vein thrombosis (DVT), we observed an unexpected difference in 6-month mortality among cancer patients in favor of LMWH, which could not be attributed to a difference in the incidence of thrombotic or bleeding complications.6 A similar observation in favor of LMWH was reported in a subsequent study and in a meta-analysis of trials.7,8 The number of cancer patients included in these studies was small, and adjustment of the observed effect for the baseline characteristics of the cancer patients was not possible. However, these findings suggested an inhibitory effect of LMWH on tumor growth or metastasis, which is less apparent or absent for UFH, resulting in a beneficial effect on the survival of cancer patients. This hypothesis is supported by the observations, in experimental studies, that LMWH and low molecular weight heparan sulfate, in comparison to UFH, effectively suppressed angiogenesis, a process necessary for tumor growth and metastasis.9,10 On the other hand, animal studies that investigated the effect of chemically-modified heparins on the spread of cancer did not detect a superior anti-tumor effect of LMWH compared to UFH; both were found to inhibit metastasis.11,12 To date, the effect of LMWH on cancer survival in humans has not been investigated as a primary objective. If a consistent and beneficial effect of LMWH on mortality is indeed present, such a study would be warranted.We performed a meta-analysis of all available randomized clinical trials where LMWH was compared with UFH in the treatment of venous thromboembolism (VTE) to estimate the crude treatment effect of LMWH on mortality in cancer patients compared to UFH. Subsequently, we adjusted this treatment effect for age, gender, and primary malignancy site by reanalyzing data from three of those trials.3-5 This effect was further adjusted for other prognostic factors, including cancer histology, tumor stage, presence of metastases, duration of cancer, and concomitant use of cancer treatment, by analyzing individual patient data from the largest randomized trial.5

“In vitro” fibrinolytic activity of a new low molecular weight heparan sulfate

The fibrinolytic activity, effect on the fibrinolytic activity of plasmin, anticoagulant activity and anti platelet aggregation activity of a low molecular weight heparan sulfate ( LMW HS Bal ) were investigated “in vitro” on blood plasma obtained from rats and rabbits. LMW HS Bal at concentrations as low as 0.25 – 2 μg/ml prevented thrombin-induced platelet aggregation. At concentrations 25 to 50 times larger it showed no significant anticoagulant activity but a marked fibrinolytic effect. At still larger concentrations LMW HS Bal also potentiated the fibrinolytic activity of plasmin. Conversely, unfractionated heparan sulfate (UHS) at concentrations of 25 to 50 μg/ml, only showed anticoagulant activity with no fibrinolytic activity.

Heparan sulfate proteoglycans of rat embryo fibroblasts. A hydrophobic form may link cytoskeleton and matrix components.

Heparan sulfate proteoglycans (HSPGs) synthesized in cultures of rat embryo fibroblasts have been isolated and characterized. Cells grown in the presence of [35S]sulfate secreted a large amount of radiolabeled macromolecules into the culture medium of which only a small proportion was identified as HSPG. However, the majority of radiolabeled proteoglycans isolated from the cell layer were HSPGs. Here, two types of HSPG were detected. One type had an Mr of 5-8 X 10(5) as estimated by gel chromatography on Sepharose CL-4B in the presence of 0.1% sodium dodecyl sulfate and lacked hydrophobic properties in that it showed no affinity for octyl-Sepharose and could not be inserted into liposomes. The other HSPG type had an estimated Mr of 3-5 X 10(5), was retained on octyl-Sepharose, and could be inserted into liposomes. In addition, the cells contained low molecular weight heparan sulfate oligosaccharides. Treatment of living cells with 0.2% Triton X-100, which retained stress fibers and extracellular matrix but solubilized cell membranes, released a proportion of the smaller HSPG together with the heparan sulfate oligosaccharides. The cytoskeleton-matrix residue remaining after detergent extraction of the cell contained the larger species of HSPG in addition to the smaller HSPG. The presence of the smaller hydrophobic HSPG in the detergent-treated cytoskeleton-matrix preparations suggests that it may form part of a transmembrane cytoskeleton-matrix linkage.

Substrate Specificity of a Heparan Sulfate-Degrading Endoglucuronidase from Human Placenta

A heparan sulfate-degrading endoglucuronidase was isolated from human placenta and partially purified by affinity chromatography on heparan sulfate-Sepharose 4B. The endoglucuronidase has a molecular weight of approximately 100 000 estimated by gel chromatography and a broad pH optimum between pH4 and pH6. Carboxyl reduced heparan sulfate is not split by partially purified endoglucuronidase, but inhibits the action of that enzyme towards non-modified heparan sulfate. Low molecular weight heparan sulfate (Mr approximately 3 000) is not attacked by the endoglucuronidase. N-Desulfated heparan sulfate and heparin are only weak substrates. The amino sugar adjacent to the glucuronic acid residue appearing at the reducing terminal of heparan sulfate fragments liberated by the endoglucuronidase appears to be exclusively N-acetylated glucosamine.