The primary prevention of CVD is dependent upon the ability to identify high-risk individuals long before the development of overt events. This highlights the need for biomarkers and accurate diagnosis. Despite an overwhelming number of individual biomarkers reported on cardiovascular disease, no one biomarker is found to be reliable in predicting or diagnosing a cardiovascular event.
The glycocalyx is an endothelial surface layer which lines the epithelial cells of the vascular system, protecting epithelial cells from vascular flow shear stress, coagulants and platelets, and leukocyte-cell interactions. Glycoproteins component of glycocalyx function as receptors on the cell surface, such as selectins, integrins, and members of the immunoglobulin superfamily; these are weaved into the net of the endothelial glycocalyx. Also, the endothelial surface layer consists of secreted proteoglycans (eg, versican and perlecan) and their adsorbed plasma proteins (eg, orosomucoid and albumin). Together with glycosaminoglycans (GAGs) and plasma proteins, the endothelial surface layer as a whole forms a dynamic barrier to circulating cells and soluble biologic macromolecules.
The Glycocalyx is key to assessing, preventing and/or treating plaque formation leading to coronary heart disease (CHD). The glycocalyx is now recognized as a key structure for maintaining vascular wall integrity. Disruption or a decrease in thickness can result in chronic vascular disease; for example chronic stagnant blood flow, common in bifurcated sections of the arteries, triggers glycocalyx shedding and plaque formation. When damaged, the glycocalyx sheds three components: syndecans, heparan sulfate, and hyaluronan. Coincident to shedding is the release of plasminogen activator inhibitor–1 which is a potent inhibitor of fibrinolysis8. Disturbance of the glycocalyx marks early stages of various clinical pathophysiologies including thrombus formation and atherscloerosis.
Studies and Results
4-Panel Pre-Clinical Study
Coronary heart disease (CHD) is a member of the cardiovascular family (CVD) and leading disease killer in the world. CHD characteristic feature is plaque formation, which results in atherosclerosis or hardening of the arteries. Plaque formation is triggered by glycocalyx disruption and the shedding of glycocalyx detritus. In this regard 4 glycocalyx detritus (GlycocardiaCHD) were selected as components of the fingerprint, namely: syndecan-1 (SDC-1), heparan SO4 (HS), hyaluroman-1 (HAS-1 :), and plasminogen activator inhibitor -1 (SDC-1). Dr. Tunac also developed the Tunac Arterial Plaque (TAP) Natural Mouse™ model, used to model plaque formation as it occurs in humans. Indeed, the blood levels of the 4 detritus correlated with plaque formation:
5-Panel Clinical Study
The correlation of blood levels of the 4 glycocalyx detritus to plaque formation prompted the evaluation of IRB clinical samples. These clinical samples represented blood drawn from patients suffering from chest pain, heart failure (HF) and hypertension (HTN);
Cardiac troponin (cTn), proteins found in skeletal and heart muscle fibers is the most common diagnostic tool in the ER. The test is ordered if a person is experiencing symptoms such as: chest pain (angina), shortness of breath (heart failure), and hypertension (rapid heart rate, lightheadedness, fatigue). Even with the widespread use of cTn assays worldwide, there remains some confusion among clinicians and laboratorians about the timing, frequency, and duration for measuring cTn after patients present with symptoms suggestive of acute coronary syndrome (ACS) highlighting the discrepancies and errors that can occur when relying on a single biomarker. For this reason, the 5 glycocalyx detritus were selected, namely: growth differentiation factor-15 (GDF), plasminogen activator inhibitor -1 (PAI-1), pregnancy associated plasma protein –A (PAPP-A), syndecan-1 (SDC-1), and heparan SO4 (HS). Blood levels of these 5 detritus were evaluated using ELISA. The levels of the 5 glycocalyx detritus were elevated in each of the diseases; moreover, each disease produced a characteristic pattern or fingerprint.
7-Panel Clinical Studies
Study 1: Serum blood (IRB) samples were drawn from patients clinically identified with chronic diseases, including coronary heart disease (CHD), heart failure (HF), rheumatoid arthritis (RA), stroke, hypertension (HTN), diabetes 2 (DIAB), Alzheimer's (ALZ) and healthy (HEALTH) individuals. Samples were analyzed per ELISA for levels of detritus.
Study 2: To establish a standard operating procedure (SOP), both serum and plasma were then evaluated. The results show that ELISA levels of the markers were generally comparable. However, no evaluable levels were found for gamma fibrinogen in the serum samples as illustrated. In this regard, plasma preparation is be the preferred sample moving forward to build an “Arterez Disease Repository”.
The observed sequence of blood marker levels detected from highest to lowest is as follows: Gamma fibrinogen (GAMMA) > heparan sulfate (HEP) > hyaluronan (HAS)> pregnancy associated plasma protein (PAP) > growth differentiation factor 15 (G15) > plasminogen activator inhibitor (PAI) > syndecan-1 (SDC).
Characteristic fingerprint for each disease:
Each disease showed a unique ‘fingerprint’, which confirms the effectiveness of the GDF as a tool for diagnosing a wide spectrum of chronic diseases
NIH SBIR Study Submissions & Aims
3-Disease Remnant Study
AIM 1: Qualify ELISA assays for quantitating levels of Heparan SO4 (HS), Pregnancy Associated Plasma Protein (PAPP-A), Growth Differentiation Factor 15 (GDF-15), Syndecan-1 (SDC-1), Plasminogen Activator Inhibitor -1 (PAI-1), Gamma Fibrinogen (FGG), and Hyaluronan Synthase -1 (HAS-1) using 250 human serum (remnant) samples with a clinical diagnosis of coronary artery disease (CAD), hypertension and heart failure.
AIM 2: We have designed a statistical process and methodology to develop the foundation of disease algorithms. This will be accomplished by using the proposed study data to arrive at a path analysis to establish causality between and amongst the biomarkers and indications, beginning with CAD. Analysis will be done in several steps to underpin the data as a system of checks and balances for each data set. Several steps will provide rapid access to demographic and other data within the patient population and will provide confirmation of predictability of the seven-panel matrix.
6 Disease IRB Pilot Study
AIM 1: Qualify ELISA assays and Mass Spec (proteomics) for quantitating levels of Heparan SO4 (HS), Pregnancy Associated Plasma Protein (PAPP-A), Growth Differentiation Factor 15 (GDF-15), Syndecan-1 (SDC-1), Plasminogen Activator Inhibitor -1 (PAI-1), Gamma Fibrinogen (FGG), and Hyaluronan Synthase -1 (HAS-1) using 21 IRB approved human serum samples (plus control group) - with a clinical diagnosis of CAD, hypertension, heart failure, diabetes type II, rheumatoid arthritis and stroke.
AIM 2: Utilize same statistical process and methodology as above to develop the foundation of disease algorithms for further ELISA focus and development across multiple vascular diseases beyond the family of CVD. This will be accomplished by using the proposed study data to arrive at a path analysis.
Companion and stand-alone diagnostic
GlycoCardia™ can be used twofold: 1) as a companion diagnostic for Embotricin™, or 2) ‘stand-alone’ diagnostic to monitor or evaluate the traditional symptom-targeted therapies for their ability to restore glycocalyx (hyaluronan, heparan, syndecan), clotting potential (PAI-1), mitigate hypertension, heart failure, stroke, and CAD. The latter presents an immediate market for GlycoCardia™.
(a) Provides specific information concerning the associated pathology and risk factors for the CVD family.
(b) Unique and proprietary blood test algorithm.
(c) Adoptable ELISA test structure; utilizes existing equipment found in both acute and chronic care settings.
(d) Proved to predict onset of clot and production of plaques in tested animals.
(e) Currently no diagnostic test available providing patient specific information concerning the pathology or the absolute risk factors for the family of CV Diseases.
Our intellectual properties include;
Drug Treatment and Biomarker Panel Targeted to Diseases Due to Multifactorial Ontology of Glycocalyx Disruption.
Biomarkers of Vascular Disease.
Methods and Compositions for Reversing Disruption of the Glycocalyx, Inflammation and Oxidative Damage.
(Draft) Animal Model for CVD and Inflammation.
Innovation Meets High Impact and Unmet Need
7 panel GDF (GlycocardiaGEN) – Clinical Data
Blood levels of 7 detritus components were obtained from published literature of patients with coronary heart disease (CHD), heart failure (HF), and hypertension and a virtual fingerprint was constructed. Each disease showed a unique fingerprint, which confirms the effectiveness of the GDF technology as a unique tool for identifying and monitoring treatments for diseases.
Introducing the Glycalyx Detritus Fingerprint (GDF)