Boguslaw Lipinski

Pathophysiology of oxidative stress in diabetes mellitus.

Oxidative stress is believed to play an important role, albeit not fully recognized, in the development of vascular complications in diabetes mellitus (DM) particularly type 2. In the majority of studies, attention was focused on lipid oxidation, specifically on that of low-density lipoproteins (LDLs). More recent investigations have revealed that it is not only the lipid but also the apolipoprotein moiety of LDL that becomes oxidatively modified resulting in the formation of insoluble aggregates. Consequently, it has been documented that LDL aggregation was due to the hydroxyl radical-induced dityrosine crosslinking between apo B monomers. In DM patients with atherosclerotic complications, intravascular fibrous deposits were shown to contain, in addition to oxidized LDL, a fibrin-like material (FLM). This material is immunologically identical to fibrin that is normally formed as a result of intravascular activation of the blood coagulation cascade. Although DM patients with vascular disease display increased concentration of plasma fibrinogen (Fbg), the precursor of fibrin, no markers of full blown activation of blood coagulation could be found.

Association of Urinary Inflammatory Markers and Renal Decline in Microalbuminuric Type 1 Diabetics

Progressive renal function decline begins in one third of patients with microalbuminuria and type 1 diabetes. This study examined whether this decline is associated with elevated excretion of inflammatory markers in urine. Five inflammatory markers (IL-6, IL-8, monocyte chemoattractant protein-1, interferon-gamma-inducible protein (IP-10), and macrophage inflammatory protein-1delta) were measured in urine samples from the First Joslin Study of the Natural History of Microalbuminuria in Type 1 Diabetes, a cohort recruited in 1991. Samples were obtained from 43 participants with microalbuminuria and stable renal function (nondecliners), from 28 with microalbuminuria and early progressive renal function decline (decliners), and from 74 with normoalbuminuria and stable renal function (reference). Urinary concentrations of all five inflammatory markers were significantly higher in decliners than in nondecliners, who were similar to the reference group. Multivariate analysis revealed that those with more than two markers elevated were more than five times as likely to have early progressive decline of renal function. In contrast, serum concentrations of C-reactive protein, IL-8, and macrophage inflammatory protein-1delta did not differ between decliners and nondecliners. These results support the hypothesis that inflammatory processes in the kidney contribute to the progression of nephropathy in patients with type 1 diabetes.

Changes in red blood cell membrane structure in type 2 diabetes: a scanning electron and atomic force microscopy study

Red blood cells (RBCs) are highly deformable and possess a robust membrane that can withstand shear force. Previous research showed that in diabetic patients, there is a changed RBC ultrastructure, where these cells are elongated and twist around spontaneously formed fibrin fibers. These changes may impact erythrocyte function. Ultrastructural analysis of RBCs in inflammatory and degenerative diseases can no longer be ignored and should form a fundamental research tool in clinical studies. Consequently, we investigated the membrane roughness and ultrastructural changes in type 2 diabetes. Atomic force microscopy (AFM) was used to study membrane roughness and we correlate this with scanning electron microscopy (SEM) to compare results of both the techniques with the RBCs of healthy individuals. We show that the combined AFM and SEM analyses of RBCs give valuable information about the disease status of patients with diabetes. Effectiveness of treatment regimes on the integrity, cell shape and roughness of RBCs may be tracked, as this cell’s health status is crucial to the overall wellness of the diabetic patient.

Profound Morphological Changes in the Erythrocytes and Fibrin Networks of Patients with Hemochromatosis or with Hyperferritinemia, and Their Normalization by Iron Chelators and Other Agents

It is well-known that individuals with increased iron levels are more prone to thrombotic diseases, mainly due to the presence of unliganded iron, and thereby the increased production of hydroxyl radicals. It is also known that erythrocytes (RBCs) may play an important role during thrombotic events. Therefore the purpose of the current study was to assess whether RBCs had an altered morphology in individuals with hereditary hemochromatosis (HH), as well as some who displayed hyperferritinemia (HF). Using scanning electron microscopy, we also assessed means by which the RBC and fibrin morphology might be normalized. An important objective was to test the hypothesis that the altered RBC morphology was due to the presence of excess unliganded iron by removing it through chelation. Very striking differences were observed, in that the erythrocytes from HH and HF individuals were distorted and had a much greater axial ratio compared to that accompanying the discoid appearance seen in the normal samples. The response to thrombin, and the appearance of a platelet-rich plasma smear, were also markedly different. These differences could largely be reversed by the iron chelator desferal and to some degree by the iron chelator clioquinol, or by the free radical trapping agents salicylate or selenite (that may themselves also be iron chelators). These findings are consistent with the view that the aberrant morphology of the HH and HF erythrocytes is caused, at least in part, by unliganded (‘free’) iron, whether derived directly via raised ferritin levels or otherwise, and that lowering it or affecting the consequences of its action may be of therapeutic benefit. The findings also bear on the question of the extent to which accepting blood donations from HH individuals may be desirable or otherwise.

A novel method for assessing the role of iron and its functional chelation in fibrin fibril formation: the use of scanning electron microscopy

Abstract Aims: Inflammatory diseases associated with iron overload are characterized by a changed coagulation profile, where there is a persistent presence of fibrin-like material of dense-matted deposits (DMDs). It is believed that one source of such material is a result of the activation of blood coagulation without the generation of thrombin, causing clots to become resistant to fibrinolytic dissolution. The aim of the current manuscript therefore is to apply a novel scanning electron microscopy method for assessing the role of functional chelation in the prevention or reversal of iron-induced fibrin formation. Methods and results: Purified fibrinogen and platelet-rich plasma were exposed to chelating agents followed by iron, to determine the chelating effects. We show that there is another, pathological pathway of fibrin formation initiated by free iron (initially as Fe (III)), leading to the formation of highly reactive oxygen species such as the hydroxyl radical that can oxidize and insolubilize proteins, a process that might be inhibited by iron-chelating compounds. The final product of such a pathway is a fibrin-like material, termed DMDs that are remarkably resistant to proteolytic degradation. Conclusions: Scanning electron microscopy shows that iron-chelating agents are effective inhibitors of DMD formation. The most active inhibitors of DMD formation proved to be Desferal, Clioquinol and Curcumin, whereas Epigallocatechin gallate and Deferiprone were less effective. The functional model we describe may point the clinical utility of various substances in iron-mediated degenerative diseases.

High ferritin levels have major effects on the morphology of erythrocytes in Alzheimer's disease

Introduction: Unliganded iron both contributes to the pathology of Alzheimers disease (AD) and also changes the morphology of erythrocytes (RBCs). We tested the hypothesis that these two facts might be linked, i.e., that the RBCs of AD individuals have a variant morphology, that might have diagnostic or prognostic value. Methods: We included a literature survey of AD and its relationships to the vascular system, followed by a laboratory study. Four different microscopy techniques were used and results statistically compared to analyze trends between high and normal serum ferritin (SF) AD individuals. Results: Light and scanning electron microscopies showed little difference between the morphologies of RBCs taken from healthy individuals and from normal SF AD individuals. By contrast, there were substantial changes in the morphology of RBCs taken from high SF AD individuals. These differences were also observed using confocal microscopy and as a significantly greater membrane stiffness (measured using force-distance curves). Conclusion: We argue that high ferritin levels may contribute to an accelerated pathology in AD. Our findings reinforce the importance of (unliganded) iron in AD, and suggest the possibility both of an early diagnosis and some means of treating or slowing down the progress of this disease.

Iron-Induced Fibrin in Cardiovascular Disease

Accumulating evidence within the last two decades indicates the association between cardiovascular disease (CVD) and chronic inflammatory state. Under normal conditions fibrin clots are gradually degraded by the fibrinolytic enzyme system, so no permanent insoluble deposits remain in the circulation. However, fibrinolytic therapy in coronary and cerebral thrombosis is ineffective unless it is installed within 3-5 hours of the onset. We have shown that trivalent iron (FeIII) initiates a hydroxyl radical-catalyzed conversion of fibrinogen into a fibrin-like polymer (parafibrin) that is remarkably resistant to the proteolytic dissolution and thus promotes its intravascular deposition. Here we suggest that the persistent presence of proteolysis-resistant fibrin clots causes chronic inflammation. We study the effects of certain amphiphilic substances on the iron- and thrombin-induced fibrinogen polymerization visualized using scanning electron microscopy. We argue that the culprit is an excessive accumulation of free iron in blood, known to be associated with CVD. The only way to prevent iron overload is by supplementation with iron chelating agents. However, administration of free radical scavengers as effective protection against persistent presence of fibrin-like deposits should also be investigated to contribute to the prevention of cardiovascular and other degenerative diseases.

Interaction of Fibrin with Red Blood Cells: The Role of Iron

Activation of coagulation pathways results in the formation of hemostatic fibrin plugs. Under normal physiologic conditions fibrin clots are gradually, albeit completely, degraded by a fibrinolytic enzyme system to ensure proper wound healing and/or blood vessel patency. Yet in pathological situations, thrombi are not effectively removed, leading to chronic thrombosis. The susceptibility of blood clots to enzymatic degradation depends on the structure and properties of fibrin fibers. Many factors have been suspected as culprits, including red blood cells (RBCs) that become transiently trapped within fibrin mesh. Here, the authors show that there is indeed a specific interaction between RBCs and fibrin-like fibers identified here as dense matted deposits (DMDs) by means of scanning electron microscopy (SEM). It is emphasized that such interactions can be observed in ischemic stroke patients, but not from healthy subjects. However, DMD/RBC aggregates can be induced in normal blood by the additions of trivalent iron ions. The plausible mechanism of the enhanced fibrin–red blood cell interaction is based on the previously described iron-induced generation of hydroxyl radicals. These radicals cause, in turn, non-enzymatic formation of fibrinogen aggregates remarkably resistant to fibrinolysis that are also similar to DMDs described in this paper. In conclusion, this relatively simple SEM analysis may become a convenient tool for diagnosing prothrombotic conditions associated with iron overload. It is suggested that future research on prevention and treatment of ischemic stroke and other thrombosis associated diseases should include testing of iron-chelating and hydroxyl radical-scavenging agents.

Iron enhances generation of fibrin fibers in human blood: Implications for pathogenesis of stroke

Stroke is associated with the intracerebral formation of fibrin clots which may lead to irreversible brain damage. Thrombolytic therapies employ a variety of natural and/or recombinant plasminogen activators to initiate fibrinolytic degradation of cerebral thrombi. However, such therapies when installed beyond 4‐ to 6‐h window, may fail to achieve the expected outcome. This is due to the hydroxyl radical‐induced modification of fibrin(ogen) molecules rendering them refractory to fibrinolytic degradation, but no cause of the increased free radical generation in stroke was offered. Here, we show by means of electron microscopy that iron ions added to human blood dramatically enhances fibrin fibers formation with thrombin, and significantly delays fibrinolysis during spontaneous clotting of native blood. Iron ions caused the appearance dense matted fibrin deposits, similar, if not identical, to those observed in plasma of patients with stroke. These results may explain a known relationship between thrombotic diseases and the increased body concentrations of free iron and/or hemoglobin derivatives. We conclude that any action resulting in the inhibition of hemostatic abnormalities, as well as in the reduction of body free iron and scavenging of hydroxyl radicals (e.g., by polyphenols) can potentially prevent pathological reactions associated with consequences of stroke. Microsc. Res. Tech. 75:1185–1190, 2012.

Oxidation Inhibits Iron-Induced Blood Coagulation

Blood coagulation under physiological conditions is activated by thrombin, which converts soluble plasma fibrinogen (FBG) into an insoluble clot. The structure of the enzymatically-generated clot is very characteristic being composed of thick fibrin fibers susceptible to the fibrinolytic degradation. However, in chronic degenerative diseases, such as atherosclerosis, diabetes mellitus, cancer, and neurological disorders, fibrin clots are very different forming dense matted deposits (DMD) that are not effectively removed and thus create a condition known as thrombosis. We have recently shown that trivalent iron (ferric ions) generates hydroxyl radicals, which subsequently convert FBG into abnormal fibrin clots in the form of DMDs. A characteristic feature of DMDs is their remarkable and permanent resistance to the enzymatic degradation. Therefore, in order to prevent thrombotic incidences in the degenerative diseases it is essential to inhibit the iron-induced generation of hydroxyl radicals. This can be achieved by the pretreatment with a direct free radical scavenger (e.g. salicylate), and as shown in this paper by the treatment with oxidizing agents such as hydrogen peroxide, methylene blue, and sodium selenite. Although the actual mechanism of this phenomenon is not yet known, it is possible that hydroxyl radicals are neutralized by their conversion to the molecular oxygen and water, thus inhibiting the formation of dense matted fibrin deposits in human blood.