Presence of hyaluronan in lung alveoli in severe Covid-19: An opening for new treatment options?

Severe coronavirus disease 2019 (Covid-19) is characterized by inflammation of the lungs with increasing respiratory impairment. In fatal Covid-19, lungs at autopsy have been filled with a clear liquid jelly. However, the nature of this finding has not yet been determined. The aim of the study was to demonstrate whether the lungs of fatal Covid-19 contain hyaluronan, as it is associated with inflammation and acute respiratory distress syndrome (ARDS) and may have the appearance of liquid jelly. Lung tissue obtained at autopsy from three deceased Covid-19 patients was processed for hyaluronan histochemistry using a direct staining method and compared with staining in normal lung tissue. Stainings confirmed that hyaluronan is obstructing alveoli with presence in exudate and plugs, as well as in thickened perialveolar interstitium. In contrast, normal lungs only showed hyaluronan in intact alveolar walls and perivascular tissue. This is the first study to confirm prominent hyaluronan exudates in the alveolar spaces of Covid-19 lungs, supporting the notion that the macromolecule is involved in ARDS caused by SARS-CoV-2. The present finding may open up new treatment options in severe Covid-19, aiming at reducing the presence and production of hyaluronan in the lungs.

Is low molecular weight hyaluronan an early indicator of disease in avian systemic sclerosis?

AIM OF THE STUDY: To further elucidate the pathogenesis of systemic sclerosis (SSc) an experimental avian model was used. The University of California at Davis line 200 (UCD-200) chickens spontaneously develop a SSc-like disease that has most features of human SSc with vascular effects, inflammation, autoimmunity, and fibrosis. The first signs of disease in UCD-200 chickens are swelling and ischemic lesions of the comb and the presence of a tissue containing high amounts of glycosaminoglycan hyaluronan (HA). The aim of this study was to evaluate inflammatory and fibrotic processes of the disease with regard to the molecular weight of HA. MATERIAL AND METHODS: Comb biopsies from UCD-200 and healthy White Leghorn (WL) chickens, as controls, at different ages were studied with the histochemical localization of HA, hyaluronidase-1 (Hyal-1), cluster of differentiation 3, immunoglobulin Y, and collagen I and III. The molecular weight distribution of HA was estimated with gas-phase electrophoretic analysis. RESULTS: At 2 days of age, HA was visualized in UCD-200 chickens at the dermal part of the comb with no simultaneous staining of Hyal-1. In adult UCD-200 chickens, the comb skin was almost totally devoid of HA compared to WL chickens of the same age. An increase of low molecular weight (LMW) HA was detected in comb tissue from UCD-200 at the age of 1 day, 1 week, 2 weeks, and 4 weeks, in contrast to adult animals. CONCLUSIONS: An early inflammatory process involving LMW HA was confirmed as a possible profibrotic process. This indicates that HA might be an important participant in the early inflammatory events of SSc in UCD-200 chickens and that the disappearance of HA in skin predisposes to fibrosis.

Dermal distribution of hyaluronan in psoriatic arthritis; coexistence of CD44, MMP3 and MMP9.

Psoriatic arthritis is a chronic systemic disease in which patients develop persistent inflammation of the skin and joints, leading to disability and joint damage. The extracellular component hyaluronan (HA) plays an important role in regulatory processes such as inflammation, wound healing and tumour progression. At any site of inflammation HA can be depolymerized to low-molecular weight fragments, which, in turn, induce an array of inflammatory mediators that can lead to chronic inflammation. This study describes the serum concentration and dermal distribution of HA, its receptor CD44 and the metalloproteinases 3 and 9 in skin biopsies from patients with different types of psoriatic arthritis. Fifty-one patients with psoriatic arthritis were included in the study and classified as oligo- or poly-arthritic PsA with and without treatment. Biopsies were obtained from both involved and non-involved skin and compared with biopsies from healthy individuals. Serum HA was analysed for estimation of the total turnover of HA. The main findings were an overall redistribution of HA in both involved and non-involved psoriatic skin and an epidermal imbalance between HA and CD44. The structurally and functionally important basement membrane zone was found to be disintegrated and devoid of HA irrespective of the type of articular involvement, treatment or skin affection.

Temporal correlation between transcriptional changes and increased synthesis of hyaluronan in experimental cardiac hypertrophy.

The role of hyaluronan in cardiac growth has become evident, previously shown by increased myocardial levels of hyaluronan in a rat model of cardiac hypertrophy. To further investigate the role of hyaluronan and regulation of its synthesis in cardiac hypertrophy, quantitative measurements of myocardial hyaluronan concentration was correlated to gene transcription in hypertrophic cardiac tissue. Factor analysis was used to study this correlation over time. A subset of differentially expressed genes was identified with a transcriptional regulation correlating to the increased synthesis of hyaluronan, suggesting a common regulatory pathway. Four transcription factors, Myc, Fos, Junb and Egr1, were also up-regulated. Furthermore, the Ace gene was up-regulated, representing increase of angiotensin II, an inducer of these transcription factors and fetal genes in cardiac hypertrophy. This demonstrates a coordinated synthesis of hyaluronan and pro-hypertrophic gene expression, regulated by immediate early genes, with angiotensin II as a possible mediator.

Progression of bone marrow fibrosis in patients with essential thrombocythemia and polycythemia vera during anagrelide treatment.

Anagrelide is a second-line option for reduction of thrombocythemia in patients with chronic myeloproliferative disorders (CMPDs). A multicenter, open, phase II study of anagrelide treatment in 60 patients during 2 yr was performed by the Swedish Myeloproliferative Disorder Study Group. Adequate bone marrow biopsies were obtained from 53 of the CMPD patients [36 essential thrombocythemia (ET), 16 polycythemia vera (PV), 1 chronic idiopathic myelofibrosis (CIMF)] before treatment and compared with biopsies from 30 healthy volunteers and 34 patients with acute myeloid leukemia (AML). Higher reticulin and hyaluronan (HYA) scores were found before anagrelide therapy in the CMPD patients than in the normal controls (p < 0.001 and p < 0.001, respectively) and AML patients (p < 0.001 and p = 0.011, respectively). At the end of the study 30 CMPD patients were still on anagrelide treatment and in 19 of these patients, all diagnosed as ET (n = 16) or PV (n = 3), pretreatment bone marrow biopsies were compared with follow-up samples. After 2 yr of anagrelide therapy the reticulin and HYA scores were significantly higher than before treatment (p = 0.02 and p = 0.002, respectively). The cellularity was significantly higher (p = 0.014), although the number of megakaryocytes did not change significantly. The increase of reticulin and HYA in the bone marrow after 2 yr of treatment with anagrelide indicated progression of fibrosis. Although anagrelide is a valuable drug for reduction of platelet levels, it seems unable to stop progression of bone marrow fibrosis and hypercellularity in ET and PV.

Hyaluronan and its receptor CD44 in the heart of newborn and adult rats.

The extracellular matrix is a dynamic space and a prerequisite for the function of cardiomyocytes. We have previously reported on the distribution of the glycosaminoglycan hyaluronan (HYA) and its cellular receptor CD44 in the vascular system. In newborn rats, HYA and its receptor were colocalized, but in the adult animals, no such colocalization was observed. Furthermore, the distribution of both HYA and CD44 differed between newborn and adult animals. In this study, the distribution of HYA and its receptor CD44 is explored in the heart. Hearts from newborn and adult rats were stained for visualization of HYA and CD44 using histochemistry and immunohistochemistry. HYA stained the interstitium of the myocardium heterogeneously. Strong staining was seen in the heart valves of both newborn and adult animals. CD44 staining was sparse in hearts from both newborn and adult animals. There are no major changes in the distribution of HYA in the myocardium during the postnatal development in contrast to the blood vessels. Thus, the structure of the interstitium does not change after birth when the heart starts to grow mainly through cardiomyocyte hypertrophy rather than hyperplasia. The abundance of HYA in the heart valves is probably related to their unique physiological properties to withstand repetitive mechanical stress.

Hyaluronan in Human Vocal Folds in Smokers and Nonsmokers-A Histochemical Study.

OBJECTIVES/HYPOTHESIS: To study the hyaluronan occurrence in human vocal folds, with special regards to gender and smoking and to discuss the implications of findings. STUDY DESIGN: This is a descriptive/morphologic study. METHODS: Sixteen cadaveric vocal folds from eight individuals between 58 and 90 years old (six women and two men) were removed and studied morphologically. Three of the individuals had been cigarette smokers. A direct method for hyaluronan histochemistry using a hyaluronan-binding protein probe (HABP) was used to visualize the polysaccharide. Five examiners performed an analysis of the intensities of hyaluronan staining, independently. RESULTS: We observed intense hyaluronan staining of the vocal folds of which those from women stained considerably stronger than those from men. Stratified squamous epithelium stained for hyaluronan in all sections, whereas respiratory epithelium only stained weakly or not at all. The highest accumulation of hyaluronan occurred subepithelially in the lamina propria, corresponding to Reinke's space. It was observed that vocal folds from smokers were more intensively stained than those from nonsmokers. CONCLUSIONS: Hyaluronan is found in all layers of the human vocal fold. Contradictory to earlier studies, hyaluronan was visualized in squamous epithelium, where it may function as an impact protector. The occurrence of hyaluronan in smokers may have implications in the development of vocal fold inflammation and tumor initiation as hyaluronan is an important molecule in these processes.

Bone marrow hyaluronan distribution in patients with acute myeloid leukemia.

Acute myeloid leukemia (AML) is a clonal disorder characterized by abnormal proliferation of myeloid cell precursors. Research has mainly focused on the cellular events, but the bone marrow matrix has attracted minor interest. In this study bone marrow biopsies were obtained from 35 newly diagnosed AML patients. The bone marrows were analyzed regarding the occurrence and distribution of hyaluronan (HYA) and reticulin fibers (type III collagen). The bone marrow sections were analyzed histochemically and compared with bone marrows from 30 healthy controls. The HYA staining was significantly stronger in the AML patients compared with the controls. Only one patient demonstrated abnormal reticulin staining score, but in the group of patients with antecedent myelodysplastic syndrome (MDS), the reticulin staining score was significantly higher compared with the patients with de novo AML. There was a significant correlation between the HYA staining and reticulin staining scores in the AML patients as was seen in the control group.

High Sensitivity Method to Estimate Distribution of Hyaluronan Molecular Sizes in Small Biological Samples Using Gas-Phase Electrophoretic Mobility Molecular Analysis.

Hyaluronan is a negatively charged polydisperse polysaccharide where both its size and tissue concentration play an important role in many physiological and pathological processes. The various functions of hyaluronan depend on its molecular size. Up to now, it has been difficult to study the role of hyaluronan in diseases with pathological changes in the extracellular matrix where availability is low or tissue samples are small. Difficulty to obtain large enough biopsies from human diseased tissue or tissue from animal models has also restricted the study of hyaluronan. In this paper, we demonstrate that gas-phase electrophoretic molecular mobility analyzer (GEMMA) can be used to estimate the distribution of hyaluronan molecular sizes in biological samples with a limited amount of hyaluronan. The low detection level of the GEMMA method allows for estimation of hyaluronan molecular sizes from different parts of small organs. Hence, the GEMMA method opens opportunity to attain a profile over the distribution of hyaluronan molecular sizes and estimate changes caused by disease or experimental conditions that has not been possible to obtain before.

The structure of the basement membrane zone differs between keloids, hypertrophic scars and normal skin: a possible background to an impaired function.

Scar tissues were collected from patients with keloids, hypertrophic scars and mature scars. Normal skin was obtained from healthy individuals. Clinical attributes were used to select which tissue to obtain but the distribution of the specific hyaluronan (HA) staining was then used for the definite classification of the various scar types. Light microscopic and ultrastructural analyses were performed with an HA-binding probe, antibodies for collagen I and III and staining for mast cells. Ultrastructural studies of keloids revealed an altered collagen structure in the dermal layers, with an abundance of collagen fibres of similar diameter in both the reticular dermis (RD) and the papillary dermis (PD) compared to normal skin. Furthermore, the keloids displayed epidermal changes, which involved the basement membrane (BM), with fewer hemidesmosomes and an altered shape of desmosomes in the entire enlarged spinous layer. The frequency of mast cells found in keloids was lower than in other scar tissues and normal skin. These alterations in epidermis could influence the hydrodynamic and cell regulatory properties of the wounded skin with impaired function and insufficient regulative capacity to hinder the ever-growing collagen tissue that is characteristic for keloids.

Hyaluronan and collagen in human hypertrophic cardiomyopathy: a morphological analysis.

Introduction. The hypertrophic cardiomyopathy (HCM) disease process is not only limited to cardiomyocyte abnormalities but also engages the extracellular matrix. Hyaluronan (HA) and its receptor CD44 are involved in cellular growth and tissue proliferation but have so far been less studied in myocardial hypertrophy. In HCM, collagens are abundant but their histological distribution and relation to hyaluronan have not been described. Material and Methods. Myocardial specimens from 5 patients with symptomatic left ventricular tract obstruction undergoing myectomy due to HCM were processed for histochemistry and immunohistochemistry. Results. HA staining was more intense in HCM patients. The histological distribution of HA was the same in patients and controls, that is, interstitial staining including the space between cardiomyocytes, in fibrous septa, and in the adventitia of intramyocardial blood vessels. CD44 was not detected in the myocardium of patients or controls. Collagen I showed the same general localisation as HA but detailed distribution differed. Conclusions. This is the first study that describes the distribution of hyaluronan in human HCM. HA staining is more intense in HCM patients but without coexpression of its receptor CD44, at least not in the chronic phase of HCM. HA and collagen I have the same localisation.

Evaluation of hyaluronan and calcifications in stenotic and regurgitant aortic valves.

OBJECTIVE: Hyaluronan (HA) is a major component of the interstitium and has been observed in normal heart valves. The function of HA in heart valves is unknown but contribution to biomechanical function has been proposed. The purpose of this investigation was to study the distribution of HA in relation to calcifications in diseased human aortic valves. METHODS: Human aortic valves were collected at aortic valve replacement, of whom nine patients had regurgitation and 13 stenotic disease. The valves were decalcified and stained for the visualisation of HA. The specimens were macroscopically evaluated for magnitude of calcification using image analysis. The microscopic amount and distribution of HA and calcifications were semiquantitatively evaluated using histochemistry. RESULTS: The overall HA staining showed an inverse relationship against the magnitude of observed valve calcifications (p<0.001) and type of disease (p=0.014). Multiple-group comparison revealed regionally reduced HA staining in diffuse and heavy calcified regions inside the valve (both p<0.001) compared with normal-structured parts of the valve. HA was concentrated on the ventricular side of the valve (p=0.002). CONCLUSIONS: The content of HA was reduced in calcified aortic valves and had a heterogeneous distribution, potentially contributing to poor valve function. HA may also be involved in the pathophysiological process in degenerative aortic stenosis.

Growth factor PDGF-BB stimulates cultured cardiomyocytes to synthesize the extracellular matrix component hyaluronan.

BACKGROUND: Hyaluronan (HA) is a glycosaminoglycan located in the interstitial space which is essential for both structural and cell regulatory functions in connective tissue. We have previously shown that HA synthesis is up-regulated in a rat model of experimental cardiac hypertrophy and that cardiac tissue utilizes two different HA synthases in the hypertrophic process. Cardiomyocytes and fibroblasts are two major cell types in heart tissue. The fibroblasts are known to produce HA, but it has been unclear if cardiomyocytes share the same feature, and whether or not the different HA synthases are activated in the different cell types. METHODOLOGY/PRINCIPAL FINDINGS: This study shows, for the first time that cardiomyocytes can produce HA. Cardiomyocytes (HL-1) and fibroblasts (NIH 3T3) were cultivated in absence or presence of the growth factors FGF2, PDGF-BB and TGFB2. HA concentration was quantified by ELISA, and the size of HA was estimated using dynamic light scattering. Cardiomyocytes synthesized HA but only when stimulated by PDGF-BB, whereas fibroblasts synthesized HA without addition of growth factors as well as when stimulated by any of the three growth factors. When fibroblasts were stimulated by the growth factors, reverse dose dependence was observed, where the highest dose induced the least amount of HA. With the exception of TGFB2, a trend of reverse dose dependence of HA size was also observed. CONCLUSIONS/SIGNIFICANCE: Co-cultivation of cardiomyocytes and fibroblasts (80%/20%) increased HA concentration far more that can be explained by HA synthesis by the two cell types separately, revealing a crosstalk between cardiomyocytes and fibroblasts that induces HA synthesis. We conclude that dynamic changes of the myocardium, such as in cardiac hypertrophy, do not depend on the cardiomyocyte alone, but are achieved when both cardiomyocytes and fibroblasts are present.

Parallel up-regulation of FGF-2 and hyaluronan during development of cardiac hypertrophy in rat.

The importance of glycosaminoglycan hyaluronan (HA) and its receptor CD44 in cell proliferation is becoming increasingly evident. Expression of the genes coding for hyaluronan synthase 1 (HAS1), HAS2, HAS3, CD44, fibroblast growth factor-2 (FGF-2), and FGF receptor-1 (FGFR-1) and the histological evidence for increases of HA and CD44 were investigated in an experimental rat model of cardiac hypertrophy. The abdominal aorta was ligated to induce cardiac hypertrophy, and mRNAs prepared from heart tissue were analyzed after 1, 6, and 42 days. The total concentration of HA was quantified, and HA and CD44 were studied histochemically. The expression of HAS1, HAS2, CD44, and FGF-2 was considerably up-regulated at days 1 and 6 and returned to basal levels after 42 days. FGFR-1 was up-regulated at day 1 but at basal levels once more at days 6 and 42. The concentration of HA significantly increased in aorta-ligated rats. Histochemical analysis showed increased expression of CD44 in hypertrophied myocardium mainly in and around the coronary arteries. These results agree well with other studies of tissue growth (malignancies and wound healing). The increase of HA, its synthases, and receptor in parallel with FGF-2 and its receptor illustrates their complicated interplay in the development of cardiac hypertrophy. The up-regulation of both HAS1 and HAS2 indicates the importance of HA production in the hypertrophic process and the possibility that HA is needed for two different purposes for the heart to be able to adapt to the increased afterload caused by aortic ligature.

Expression of the CD44 receptor in the blood vessel system: an experimental study in rat.

The CD44 receptor is a transmembrane glycoprotein expressed on a variety of cells like endothelial, epithelial and smooth muscle cells. This molecule has many important functions, e.g. in cell-cell and cell-matrix interactions and signal transduction. The main ligand for CD44 is hyaluronan (HYA). HYA is a glycosaminoglycan with structural and cell biological properties. The localization of HYA in the vessel wall of arteries and veins in the healthy adult and newborn rat has been described earlier. In this study the occurrence of the CD44 receptor was investigated in the same vessels and compared to the localization of HYA. Both CD44 and its ligand showed an increased expression in the vessel wall of newborn rats compared to that of adult rats. Although HYA is abundant in the adventitia of adult rats, virtually no expression of CD44 was observed. Our results indicate that the CD44 receptor expression is increased during the stage of maturation of the vessel tree whereas the CD44 receptor is less needed by HYA in the healthy vessel wall.

Hyaluronan is differently located in arteries and veins. An immunohistochemical study in the rat.

The matrix components of the vessel wall are of great importance for the function of the vessel system of which both endurance and elasticity are prerequisites. One component of the vessel wall is the glycosaminoglycan hyaluronan (HYA) with its unique physico-chemical properties, e.g. viscoelasticity and barrier function. The present study aimed to map and compare the normal localisation and distribution of hyaluronan in the arteries and veins of both adult and newborn rats, using a specific staining method utilizing a hyaluronan-binding protein. The hyaluronan stained clearly different in veins and arteries both in newborn and adult rats. In the veins, the tunica intima stained intensely whereas the corresponding area in the arteries only showed a weak and scattered staining pattern. In the adult rats, the matrix between the smooth muscle cells of the tunica media of the veins had a clearly positive staining pattern compared to the media in the arteries which showed only a few scattered areas of positive staining. In the newborn rats, the media of the arteries stained more intensely. In both newborn and adult rats, the adventitia stained intensely both in veins and arteries. Moreover, the HYA-staining pattern differed in veins in different regions of the body. Since HYA is known to be involved in many different biological processes, the results are of importance to understand physiological properties of the vessel tree and to explain the development of vascular diseases.