Damp building moulds: Assessment of sensitization in patients and studies into mechanisms of airway inflammation using experimental models

Exposure to water-damaged buildings and the associated health problems have evoked concern and created confusion during the past 20 years. Individuals exposed to moisture problem buildings report adverse health effects such as non-specific respiratory symptoms. Microbes, especially fungi, growing on the damp material have been considered as potential sources of the health problems encountered in these buildings. Fungi and their airborne fungal spores contain allergens and secondary metabolites which may trigger allergic as well as inflammatory types of responses in the eyes and airways. Although epidemiological studies have revealed an association between damp buildings and health problems, no direct cause-and-effect relationship has been established. Further knowledge is needed about the epidemiology and the mechanisms leading to the symptoms associated with exposure to fungi. Two different approaches have been used in this thesis in order to investigate the diverse health effects associated with exposure to moulds. In the first part, sensitization to moulds was evaluated and potential cross-reactivity studied in patients attending a hospital for suspected allergy. In the second part, one typical mould known to be found in water-damaged buildings and to produce toxic secondary metabolites was used to study the airway responses in an experimental model. Exposure studies were performed on both naive and allergen sensitized mice. The first part of the study showed that mould allergy is rare and highly dependent on the atopic status of the examined individual. The prevalence of sensitization was 2.7% to Cladosporium herbarum and 2.8% to Alternaria alternata in patients, the majority of whom were atopic subjects. Some of the patients sensitized to mould suffered from atopic eczema. Frequently the patients were observed to possess specific serum IgE antibodies to a yeast present in the normal skin flora, Pityrosporum ovale. In some of these patients, the IgE binding was partly found to be due to binding to shared glycoproteins in the mould and yeast allergen extracts. The second part of the study revealed that exposure to Stachybotrys chartarum spores induced an airway inflammation in the lungs of mice. The inflammation was characterized by an influx of inflammatory cells, mainly neutrophils and lymphocytes, into the lungs but with almost no differences in airway responses seen between the satratoxin producing and non-satratoxin producing strain. On the other hand, when mice were exposed to S. chartarum and sensitized/challenged with ovalbumin the extent of the inflammation was markedly enhanced. A synergistic increase in the numbers of inflammatory cells was seen in BAL and severe inflammation was observed in the histological lung sections. In conclusion, the results in this thesis imply that exposure to moulds in water damaged buildings may trigger health effects in susceptible individuals. The symptoms can rarely be explained by IgE mediated allergy to moulds. Other non-allergic mechanisms seem to be involved. Stachybotrys chartarum is one of the moulds potentially responsible for health problems. In this thesis, new reaction models for the airway inflammation induced by S. chartarum have been found using experimental approaches. The immunological status played an important role in the airway inflammation, enhancing the effects of mould exposure. The results imply that sensitized individuals may be more susceptible to exposure to moulds than non-sensitized individuals.

The Pathobiology of the Saccular Cerebral Artery Aneurysm Rupture and Repair : A Clinicopathological and Experimental Approach

Backround and Purpose The often fatal (in 50-35%) subarachnoid hemorrhage (SAH) caused by saccular cerebral artery aneurysm (SCAA) rupture affects mainly the working aged population. The incidence of SAH is 10-11 / 100 000 in Western countries and twice as high in Finland and Japan. The estimated prevalence of SCAAs is around 2%. Many of those never rupture. Currently there are, however, no diagnostic methods to identify rupture-prone SCAAs from quiescent, (dormant) ones. Finding diagnostic markers for rupture-prone SCAAs is of primary importance since a SCAA rupture has such a sinister outcome, and all current treatment modalities are associated with morbidity and mortality. Also the therapies that prevent SCAA rupture need to be developed to as minimally invasive as possible. Although the clinical risk factors for SCAA rupture have been extensively studied and documented in large patient series, the cellular and molecular mechanisms how these risk factors lead to SCAA wall rupture remain incompletely known. Elucidation of the molecular and cellular pathobiology of the SCAA wall is needed in order to develop i) novel diagnostic tools that could identify rupture-prone SCAAs or patients at risk of SAH, and to ii) develop novel biological therapies that prevent SCAA wall rupture. Materials and Methods In this study, histological samples from unruptured and ruptured SCAAs and plasma samples from SCAA carriers were compared in order to identify structural changes, cell populations, growth factor receptors, or other molecular markers that would associate with SCAA wall rupture. In addition, experimental saccular aneurysm models and experimental models of mechanical vascular injury were used to study the cellular mechanisms of scar formation in the arterial wall, and the adaptation of the arterial wall to increased mechanical stress. Results and Interpretation Inflammation and degeneration of the SCAA wall, namely loss of mural cells and degradation of the wall matrix, were found to associate with rupture. Unruptured SCAA walls had structural resemblance with pads of myointimal hyperplasia or so called neointima that characterizes early atherosclerotic lesions, and is the repair and adaptation mechanism of the arterial wall after injury or increased mechanical stress. As in pads of myointimal hyperplasia elsewhere in the vasculature, oxidated LDL was found in the SCAA walls. Immunity against OxLDL was demonstrated in SAH patients with detection of circulating anti-oxidized LDL antibodies, which were significantly associated with the risk of rupture in patients with solitary SCAAs. Growth factor receptors associated with arterial wall remodeling and angiogenesis were more expressed in ruptured SCAA walls. In experimental saccular aneurysm models, capillary growth, arterial wall remodeling and neointima formation were found. The neointimal cells were shown to originate from the experimental aneurysm wall with minor contribution from the adjacent artery, and a negligible contribution of bone marrow-derived neointimal cells. Since loss of mural cells characterizes ruptured human SCAAs and likely impairs the adaptation and repair mechanism of ruptured or rupture-prone SCAAs, we investigated also the hypothesis that bone marrow-derived or circulating neointimal precursor cells could be used to enhance neointima formation and compensate the impaired repair capacity in ruptured SCAA walls. However, significant contribution of bone marrow cells or circulating mononuclear cells to neointima formation was not found.

Molecular mechanisms of hypertension-induced heart failure : Experimental studies with special emphasis on local renin-angiotensin system, cardiac metabolism and levosimendan

Hypertension is a major risk factor for stroke, ischaemic heart disease, and the development of heart failure. Hypertension-induced heart failure is usually preceded by the development of left ventricular hypertrophy (LVH), which represents an adaptive and compensatory response to the increased cardiac workload. Biomechanical stress and neurohumoral activation are the most important triggers of pathologic hypertrophy and the transition of cardiac hypertrophy to heart failure. Non-clinical and clinical studies have also revealed derangements of energy metabolism in hypertensive heart failure. The goal of this study was to investigate in experimental models the molecular mechanisms and signalling pathways involved in hypertension-induced heart failure with special emphasis on local renin-angiotensin-aldosterone system (RAAS), cardiac metabolism, and calcium sensitizers, a novel class of inotropic agents used currently in the treatment of acute decompensated heart failure. Two different animal models of hypertensive heart failure were used in the present study, i.e. hypertensive and salt-sensitive Dahl/Rapp rats on a high salt diet (a salt-sensitive model of hypertensive heart failure) and double transgenic rats (dTGR) harboring human renin and human angiotensinogen genes (a transgenic model of hypertensive heart failure with increased local RAAS activity). The influence of angiotensin II (Ang II) on cardiac substrate utilization and cardiac metabolomic profile was investigated by using gas chromatography coupled to time-of-flight mass spectrometry to detect 247 intermediary metabolites. It was found that Ang II could alter cardiac metabolomics both in normotensive and hypertensive rats in an Ang II receptor type 1 (AT1)-dependent manner. A distinct substrate use from fatty acid oxidation towards glycolysis was found in dTGR. Altered cardiac substrate utilization in dTGR was associated with mitochondrial dysfunction. Cardiac expression of the redox-sensitive metabolic sensor sirtuin1 (SIRT1) was increased in dTGR. Resveratrol supplementation prevented cardiovascular mortality and ameliorated Ang II-induced cardiac remodeling in dTGR via blood pressure-dependent pathways and mechanisms linked to increased mitochondrial biogenesis. Resveratrol dose-dependently increased SIRT1 activity in vitro. Oral levosimendan treatment was also found to improve survival and systolic function in dTGR via blood pressure-independent mechanisms, and ameliorate Ang II-induced coronary and cardiomyocyte damage. Finally, using Dahl/Rapp rats it was demonstrated that oral levosimendan as well as the AT1 receptor antagonist valsartan improved survival and prevented cardiac remodeling. The beneficial effects of levosimendan were associated with improved diastolic function without significantly improved systolic changes. These positive effects were potentiated when the drug combination was administered. In conclusion, the present study points to an important role for local RAAS in the pathophysiology of hypertension-induced heart failure as well as its involvement as a regulator of cardiac substrate utilization and mitochondrial function. Our findings suggest a therapeutic role for natural polyphenol resveratrol and calcium sensitizer, levosimendan, and the novel drug combination of valsartan and levosimendan, in prevention of hypertension-induced heart failure. The present study also provides a better understanding of the pathophysiology of hypertension-induced heart failure, and may help identify potential targets for novel therapeutic interventions.

Milk casein-derived tripeptides : A special focus on blood pressure and vascular function

Increased consumption of low-fat milk products is inversely associated with the risk of hypertension. The beneficial effect of milk on blood pressure is attributed to high calcium and potassium content but also to specific peptide sequences, which are cleaved from milk protein during gastrointestinal digestion, fermentation of milk with proteolytic starter cultures or enzymatic hydrolysis. Milk products fermented with Lactobacillus helveticus contain casein-derived tripeptides isoleucine-proline-proline (Ile-Pro-Pro) and valine-proline-proline (Val-Pro-Pro), which have been shown to possess antihypertensive effects in humans and in experimental animals. The aim of the present series of studies was to investigate the effects of tripeptides Ile-Pro-Pro and Val-Pro-Pro or fermented milk products containing them on vascular function and blood pressure and to elucidate the mechanisms behind them by using different experimental models of hypertension. Another aim was to characterize the acute effects of tripeptides on blood pressure and arterial stiffness in mildly hypertensive humans. Ile-Pro-Pro and Val-Pro-Pro or fermented milk products containing them attenuated the development of hypertension in two experimental models of hypertension, spontaneously hypertensive rat (SHR) and type 2 diabetic Goto-Kakizaki (GK) rat fed with high-salt diet. Significant differences in systolic blood pressure (SBP) were seen after 8 weeks treatment with tripeptide-containing products compared to control product. Plant sterols did not enhance this effect. Two differently produced tripeptide powders produced a similar attenuating effect on SBP in SHR. In mildly hypertensive subjects, a single administration of tripeptide- and plant sterol-containing fermented milk product decreased both SBP and diastolic blood pressure (DBP) over a period of 8 hours. Protective effect of tripeptides Ile-Pro-Pro and Val-Pro-Pro and fermented milk products containing them on vascular function was demonstrated in in vitro studies and long-term experimental studies. The effect was shown to be endothelium-dependent and possibly involving endothelium-derived hyperpolarizing factor (EDHF). In the clinical study, single administration of tripeptide-containing fermented milk product did not affect measures of arterial stiffness. Long-term treatment with fermented milk product containing Ile-Pro-Pro and Val-Pro-Pro inhibited angiotensin-converting enzyme (ACE) and decreased aldosterone levels thus showing beneficial effects on the renin-angiotensin system (RAS) in SHR and GK. No changes in the components of RAS were observed by the single administration of the same product in mildly hypertensive subjects. Increased levels of cGMP, NOx and citrulline suggest increased nitric oxide (NO) production by the tripeptides. Taken together, Ile-Pro-Pro and Val-Pro-Pro -containing products attenuate the development of hypertension after long-term treatment in experimental models of hypertension and possess an acute antihypertensive effect in mildly hypertensive subjects. In addition, these tripeptides show endothelium-mediated beneficial effects on vascular function. Attenuation of blood pressure increase by the tripeptides in experimental animals involves RAS, but its role in the antihypertensive effect in humans remains to be elucidated.

Xanthine oxidoreductase in essential hypertension and metabolic syndrome : Experimental studies on rodent models

Hypertension, obesity, dyslipidemia and dysglycemia constitute metabolic syndrome, a major public health concern, which is associated with cardiovascular mortality. High dietary salt (NaCl) is the most important dietary risk factor for elevated blood pressure. The kidney has a major role in salt-sensitive hypertension and is vulnerable to harmful effects of increased blood pressure. Elevated serum urate is a common finding in these disorders. While dysregulation of urate excretion is associated with cardiovascular diseases, present studies aimed to clarify the role of xanthine oxidoreductase (XOR), i.e. xanthine dehydrogenase (XDH) and its post-translational isoform xanthine oxidase (XO), in cardiovascular diseases. XOR yields urate from hypoxanthine and xanthine. Low oxygen levels upregulate XOR in addition to other factors. In present studies higher renal XOR activity was found in hypertension-prone rats than in the controls. Furthermore, NaCl intake increased renal XOR dose-dependently. To clarify whether XOR has any causal role in hypertension, rats were kept on NaCl diets for different periods of time, with or without a XOR inhibitor, allopurinol. While allopurinol did not alleviate hypertension, it prevented left ventricular and renal hypertrophy. Nitric oxide synthases (NOS) produce nitric oxide (NO), which mediates vasodilatation. A paucity of NO, produced by NOS inhibition, aggravated hypertension and induced renal XOR, whereas NO generating drug, alleviated salt-induced hypertension without changes in renal XOR. Zucker fa/fa rat is an animal model of metabolic syndrome. These rats developed substantial obesity and modest hypertension and showed increased hepatic and renal XOR activities. XOR was modified by diet and antihypertensive treatment. Cyclosporine (CsA) is a fungal peptide and one of the first-line immunosuppressive drugs used in the management of organ transplantation. Nephrotoxicity ensue high doses resulting in hypertension and limit CsA use. CsA increased renal XO substantially in salt-sensitive rats on a high NaCl diet, indicating a possible role for this reactive oxygen species generating isoform in CsA nephrotoxicity. Renal hypoxia, common to these rodent models of hypertension and obesity, is one of the plausible XOR inducing factors. Although XOR inhibition did not prevent hypertension, present experimental data indicate that XOR plays a role in the pathology of salt-induced cardiac and renal hypertrophy.

Effects of oral calcium sensitizers on experimental heart failure

Suun kautta annosteltava kalsiumherkistäjä parantaa sydämen vajaatoimintaan liittyvää pumppausvajetta kokeellisissa sydämen vajaatoimintamalleissa Huolimatta viime vuosikymmenien lääketieteellisestä kehityksestä krooninen sydämen vajaatoiminta on silti edelleen vakava, elämänlaatua voimakkaasti rajoittava sairaus. Kalsiumherkistäjät ovat uusi, sydämen pumppausvoimaa lisäävä lääkeryhmä. Levosimendaani, kotimaista alkuperää oleva kalsiumherkistäjä, on kliinisessä käytössä akuutin vajaatoiminnan hoitoon suonensisäisesti ja lyhytaikaisesti annosteltavana valmisteena. Levosimendaanilla on aktiivinen metaboliitti, OR-1896, jonka oletetaan olevan vuorokauden mittaisen levosimendaani-infuusion jälkeen havaittujen useita päiviä kestävien hyödyllisisten vaikutuksisten takana. Levosimendaanin kroonisen, suun kautta tapahtuvan annostelun vaikutuksista tieto on vähäisempää, mutta sillä näyttää olevan positiivisia vaikutuksia potilaiden raportoimana. FM Marjut Louhelainen on selvittänyt väitöskirjassaan suun kautta annosteltavan levosimendaanin ja sen pitkäkestoisen aktiivisen metaboliitin vaikutuksia kroonisen vajaatoiminnan hoidossa käyttämällä sekä hypertensiivisen sydäntaudin että 2 tyypin diabeteksen komplisoimaan sydäninfarktin kokeellisia malleja. Tutkimuksessa selvitettiin lisäksi vajaatoimintaan johtavia molekyylitason tapahtumia sydänlihaksessa. Tutkimuksessa osoitettiin, että krooninen suun kautta annosteltu hoito sekä kalsiumherkistäjä levosimendaanilla että sen aktiivisella metaboliitilla estää hypertensiiviseen sydämen vajaatoiminnan aikaasaamaa sydämen uudelleenmuovaantumista ja siihen liittyvää kuolleisuutta. Nämä vaikutukset välittyivät vähentyneen sydänlihassoluhypertrofian, solukuolleisuuden ja neurohumaraalisen aktivaation kautta. Levosimendaanin ja OR-1896:n osoitettiin myös parantavan sydämen pumppausfunktiota tyyppi 2 diabeteksen komplisoimassa sydäninfarktissa. Ei-diabeettiseen tilanteeseen verrattuna diabetekseen liittyvä infarktin jälkeinen vajaatoiminnan kehitys oli yhteydessä lisääntyneeseen tulehdukseen, fibroosiin, solukuolemaan, neurohumoraaliseen aktivaatioon ja ennenaikaiseen kudoksen vanhenemiseen. Sekä levosimendaani, että OR-1869 vähensivät tulehduksen, fibroosin ja solukuoleman merkkejä ja vaimensi neurohumoraalista aktivaatiota. OR-1896 myös vähensi solujen vanhenemiseen liittyvien merkkiaineiden ilmentymistä. Väitöskirjassa todettiin, että suun kautta annosteltuna sekä levosimendaani, että sen aktiivinen metaboliitti OR-1896, omaavat terapeuttista potentiaalia sekä hypertensiivisen sydäntaudin hoitoon että sydäninfarktin jälkeisen vajaatoiminnan estoon. FM Marjut Louhelaisen farmakologian alaan kuuluva väitöskirja Effects of oral calcium sensitizers on experimental heart failure tarkastetaan Helsingin yliopiston Lääketieteellisessä tiedekunnassa perjantaina 29.01.2010 klo 12 (Biomedicum Helsinki, luentosali 2, Haartmaninkatu 8, Helsinki). Vastaväittäjänä toimii professori Raimo Tuominen, Helsingin yliopiston Farmasian tiedekunnasta ja kustoksena professori Eero Mervaala Helsingin yliopiston Lääketieteellisestä tiedekunnasta.

Expression and function of angiotensins in the regulation of intraocular pressure - an experimental study

Glaucoma is a multifactorial long-term ocular neuropathy associated with progressive loss of the visual field, retinal nerve fiber structural abnormalities and optic disc changes. Like arterial hypertension it is usually a symptomless disease, but if left untreated leads to visual disability and eventual blindness. All therapies currently used aim to lower intraocular pressure (IOP) in order to minimize cell death. Drugs with new mechanisms of action could protect glaucomatous eyes against blindness. Renin-angiotensin system (RAS) is known to regulate systemic blood pressure and compounds acting on it are in wide clinical use in the treatment of hypertension and heart failure but not yet in ophthalmological use. There are only few previous studies concerning intraocular RAS, though evidence is accumulating that drugs antagonizing RAS can also lower IOP, the only treatable risk factor in glaucoma. The main aim of this experimental study was to clarify the expression of the renin-angiotensin system in the eye tissues and to test its potential oculohypotensive effects and mechanisms. In addition, the possible relationship between the development of hypertension and IOP was evaluated in animal models. In conclusion, a novel angiotensin receptor type (Mas), as well as ACE2 enzyme- producing agonists for Mas, were described for the first time in the eye structures participating in the regulation of IOP. In addition, a Mas receptor agonist significantly reduced even normal IOP. The effect was abolished by a specific receptor antagonist. Intraocular, local RAS would thus to be involved in the regulation of IOP, probably even more in pathological conditions such as glaucoma though there was no unambiguous relationship between arterial and ocular hypertension. The findings suggest the potential as antiglaucomatous drugs of agents which increase ACE2 activity and the formation of angiotensin (1-7), or activate Mas receptors.

New molecular strategies for prevention of fibroproliferative vascular disease : An experimental approach to restenosis

Vascular intimal hyperplasia is a major complication following angioplasty. The hallmark feature of this disorder is accumulation of dedifferentiated smooth muscle cells (SMCs) to the luminal side of the injured artery, cellular proliferation, migration, and synthesis of extracellular matrix. This finally results in intimal hyperplasia, which is currently considered an untreatable condition. According to current knowledge, a major part of neointimal cells derive from circulating precursor cells. This has outdated the traditional in vitro cell culture methods of studying neointimal cell migration and proliferation using cultured medial SMCs. Somatostatin and some of its analogs with different selectivity for the five somatostatin receptors (sst1 through sst5) have been shown to have vasculoprotective properties in animal studies. However, clinical trials using analogs selective for sst2/sst3/sst5 to prevent restenosis after percutaneous transluminal coronary angioplasty (PTCA) have failed to show any major benefits. Sirolimus is a cell cycle inhibitor that has been suggested to act synergistically with the protein-tyrosine kinase inhibitor imatinib to inhibit intimal hyperplasia in rat already at well-tolerated submaximal oral doses. The mechanisms behind this synergy and its long-term efficacy are not known. The aim of this study was to set up an ex vivo vascular explant culture model to measure neointimal cell activity without excluding the participation of circulating progenitor cells. Furthermore, two novel potential vasculoprotective treatment strategies were evaluated in detail in rat models of intimal hyperplasia and in the ex vivo explant model: sst1/sst4-selective somatostatin receptor analogs and combination treatment with sirolimus and imatinib. This study shows how whole vessel explants can be used to study the kinetics of neointimal cells and their progenitors, and to evaluate the anti-migratory and anti-proliferative properties of potential vasculoprotective compounds. It also shows how the influx of neointimal progenitor cells occurs already during the first days after vascular injury, how the contribution of cell migration is more important in the injury response than cell proliferation, and how the adventitia actively contribute in vascular repair. The vasculoprotective effect of somatostatin is mediated preferentially through sst4, and through inhibition of cell migration rather than of proliferation, which may explain why sst2/sst3/sst5-selective analogs have failed in clinical trials. Furthermore, a brief early oral treatment with the combination of sirolimus and imatinib at submaximal doses results in long-term synergistic suppression of intimal hyperplasia. The synergy is a result of inhibition of post-operative thrombocytosis and leukocytosis, inhibition of neointimal cell migration to the injury-site, and maintenance of cell integrity by inhibition of apoptosis and SMC dedifferentiation. In conclusion, the influx of progenitor cells already during the first days after injury and the high neointimal cell migratory activity underlines the importance of early therapeutic intervention with anti-migratory compounds to prevent neointimal hyperplasia. Sst4-selective analogs and the combination therapy with sirolimus and imatinib represent potential targets for the development of such vasculoprotective therapies.