Clinical, structural, biochemical and X-ray crystallographic correlates of pathogenicity for variants in the C-propeptide region of the COL3A1 gene.

Vascular Ehlers-Danlos syndrome (vEDS) is a heritable disorder of connective tissue caused by pathological variants in the COL3A1 gene, which encodes the α1 chain of type III collagen. Type III collagen is a major component of skin, arterial walls, and the gastrointestinal tract. Collagen III protein deficiency manifests as an increased risk of rupture, perforation, and dissection of these structures. The most disruptive gene variants affect the collagen helix via glycine substitutions or splice donor site mutations. The C-propeptide region of COL3A1 includes exons 49-52 and has a crucial role in initiating the C-terminal assembly of procollagen monomers in the early stages of collagen biosynthesis. Nineteen COL3A1 variants have previously been reported in these exons, of which four were associated with a severe vEDS phenotype. We identified two novel C-propeptide missense variants; p.Pro1440Leu, p.Arg1432Leu, and a non-stop mutation, c.4400A > T, p. (*1467Leuext*45). These variants produce variable phenotypes ranging from obvious acrogeria to classical or hypermobile EDS. A previously reported variant p.Lys1313Arg is of unknown clinical significance but likely benign, based on this study. Assigning disease pathogenicity remains complex, clinical phenotyping and crystal structure evidence being crucial. We briefly compare reported phenotypes for patients with missense variants in the C-propeptide domain for other human collagen disorders including COL1A1 and COL1A2 (osteogenesis imperfecta).

Four patients with Sillence type I osteogenesis imperfecta and mild bone fragility, complicated by left ventricular cardiac valvular disease and cardiac tissue fragility caused by type I collagen mutations.

Osteogenesis imperfecta (OI) type I is a hereditary disorder of connective tissue (HDCT) characterized by blue or gray sclerae, variable short stature, dentinogenesis imperfecta, hearing loss, and recurrent fractures from infancy. We present four examples of OI type I complicated by valvular heart disease and associated with tissue fragility. The diagnosis of a type I collagen disorder was confirmed by abnormal COL1A1 or COL1A2 gene sequencing. One patient was investigated with electrophoresis of collagens from cultured skin fibroblasts, showing structurally abnormal collagen type I, skin biopsy showed unusual histology and abnormal collagen fibril ultra-structure at electron microscopy. The combined clinical, surgical, histological, ultra-structural, and molecular genetic data suggest the type I collagen defect as contributory to cardiac valvular disease. The degree of tissue fragility experienced at cardiac surgery in these individuals, also reported in a small number of similar case reports, suggests that patients with OI type I need careful pre-operative assessment and consideration of the risks and benefits of cardiac surgery.

Vasorelaxing effects and inhibition of nitric oxide in macrophages by new iron-containing carbon monoxide-releasing molecules (CO-RMs).

Carbon monoxide-releasing molecules (CO-RMs) are a class of organometallo carbonyl complexes capable of delivering controlled quantities of CO gas to cells and tissues thus exerting a broad spectrum of pharmacological effects. Here we report on the chemical synthesis, CO releasing properties, cytotoxicity profile and pharmacological activities of four novel structurally related iron-allyl carbonyls. The major difference among the new CO-RMs tested was that three compounds (CORM-307, CORM-308 and CORM-314) were soluble in dimethylsulfoxide (DMSO), whereas a fourth one (CORM-319) was rendered water-soluble by reacting the iron-carbonyl with hydrogen tetrafluoroborate. We found that despite the fact all compounds liberated CO, CO-RMs soluble in DMSO caused a more pronounced toxic effect both in vascular and inflammatory cells as well as in isolated vessels. More specifically, iron carbonyls soluble in DMSO released CO with a fast kinetic and displayed a marked cytotoxic effect in smooth muscle cells and RAW 247.6 macrophages despite exerting a rapid and pronounced vasorelaxation ex vivo. In contrast, CORM-319 that is soluble in water and liberated CO with a slower rate, preserved smooth muscle cell viability, relaxed aortic tissue and exerted a significant anti-inflammatory effect in macrophages challenged with endotoxin. These data suggest that iron carbonyls can be used as scaffolds for the design and synthesis of pharmacologically active CO-RMs and indicate that increasing water solubility and controlling the rate of CO release are important parameters for limiting their potential toxic effects.

CORM-3, a carbon monoxide-releasing molecule, alters the inflammatory response and reduces brain damage in a rat model of hemorrhagic stroke.

OBJECTIVE: Intracerebral hemorrhage is accompanied by a pronounced inflammatory response that mediates brain damage but is also essential for the tissue reparative process. We assessed the effect of CORM-3, a water-soluble carbon monoxide-releasing molecule possessing anti-inflammatory properties, on inflammation and brain injury after intracerebral hemorrhage. DESIGN: In vivo and in vitro laboratory study. SETTING: Research laboratory. SUBJECTS: Male Sprague-Dawley rats, 250-350 g. INTERVENTIONS: A model of collagenase injection (2 µL) in the brain was established to induce intracerebral hemorrhage. CORM-3 (4 or 8 mg/kg) was administered intravenously at different times as follows: 1) 5 mins before collagenase; 2) 3 hrs after collagenase; and 3) 3 days after collagenase challenge. MEASUREMENTS AND MAIN RESULTS: Saline was used as a negative control. Brain damage, brain water content, and behavioral assessment were evaluated. The inflammatory response was determined at set intervals after intracerebral hemorrhage by counting peripheral neutrophils and lymphocytes, neutrophils, and activated microglia/macrophages in the intracerebral hemorrhage area and measuring plasma tumor necrosis factor-á levels. BV2 microglia and DI-TNC1 astrocytes were exposed to triton (1%) or CORM-3 (10-100 ìM) and cytotoxicity (lactic dehydrogenase assay) measured at 24 hrs. A challenge with collagenase to induce intracerebral hemorrhage caused marked brain damage and modified the levels of inflammatory markers. Pretreatment with CORM-3 significantly prevented injury, modulated inflammation, and reduced plasma tumor necrosis factor-α. CORM-3 given 3 hrs after collagenase significantly increased brain injury and tumor necrosis factor-α production. In contrast, CORM-3 given 3 days after collagenase afforded partial protection, modulated inflammation, and decreased tumor necrosis factor-α starting from the day of application. No dose-dependent effects were observed. CONCLUSIONS: CORM-3 promotes neuroprotection or neurotoxicity after intracerebral hemorrhage depending on the time of administration. Beneficial effects are achieved when CORM-3 is given either before or 3 days after intracerebral hemorrhage, namely, as a prophylactic agent or during the postacute inflammatory phase.

Relationship between leukocyte kinetics and behavioral tests changes in the inflammatory process of hemorrhagic stroke recovery.

In this study, we investigated the inflammatory response to hemorrhagic stroke (HS) as the main mechanism of brain functional recovery. Sprague-Dawley rats (n = 24) underwent surgery with sterile saline (control group, n = 12) and collagenase IV-S (stroke group, n = 12) being injected into the right striatum. White blood cell analysis, histological and immunohistological examination of the brain slices, as well as densitometric analysis of polymorphonuclear and microglial cells/macrophages were correlated with behavioral tests, and the data were subjected to appropriate statistical processing. The results indicate a strong correlation between polymorphonuclear and mononuclear changes in the blood and the zone of hemorrhagic stroke with behavioral tests of functional brain recovery. We propose that the inflammatory response is determined by kinetics of polymorphonuclear and mononuclear cells in both the blood and the hemorrhagic stroke zone. Kinetics of these cells is followed by the restoration of functions, and the maximum functional recovery is observed by the time polymorphonuclear and mononuclear stages have completed. With the development of inflammation and leukocyte kinetics, it is possible to predict functional recovery of hemorrhagic stroke. Improvement of the degree and rate of hemorrhagic stroke functional recovery may be achieved by therapeutic interventions into the inflammatory mechanisms influencing polymorphonuclear and mononuclear cell kinetics.

Structure-activity relationships of methoxychalcones as inducers of heme oxygenase-1.

Chalcones are naturally occurring flavonoids composed of two aromatic rings connected by a three-carbon unit forming an alpha-beta unsaturated carbonyl group. They are pharmacologically relevant because of their ability to exert anticarcinogenic, antimicrobial, and anti-inflammatory activities. Recent evidence indicates that the bioactivity of hydroxy-chalcones is correlated with their intrinsic property to induce the antioxidant and cytoprotective enzyme heme oxygenase-1 (HO-1). In the present study, we assessed how the methoxy substituents positioned on the two aromatic rings affect the anti-inflammatory action of different chalcones in relation to their ability to increase heme oxygenase in RAW246.7 macrophages. Structure-activity relationships of methoxychlacones were qualitatively and quantitatively examined and correlated with inhibition of endotoxin-mediated nitrite production and cytotoxic effects. Our data indicate that (i) a progressive increase in heme oxygenase activity is obtained by sequentially increasing the number of methoxy substituents in the 3,4,5- and 3',4',5'-positions of the aromatic rings; (ii) methoxy substituents placed either in the 2,4,6-positions or alone in the 4- or 4'-position are ineffective; (iii) increased heme oxygenase activity by chalcones is lost when the alpha-beta double bond and the carbonyl group are reduced or protected; (iv) the anti-inflammatory activity and cytotoxicity profiles of the chalcones examined correlate with their potency as HO-1 inducers; and (v) chalcone-mediated HO-1 induction is reduced by thiols or inhibitors of phosphatidylinositol-3 kinase (PI3K) pathway. This study provides additional information on the structural features that methoxychalcones and natural antioxidants need to possess to be considered as therapeutic agents for maximizing HO-1 expression and activity.

CORM-A1: a new pharmacologically active carbon monoxide-releasing molecule.

Carbon monoxide (CO) is emerging as an important and versatile mediator of physiological processes to the extent that treatment of animals with exogenous CO gas has beneficial effects in a range of vascular- and inflammatory-related disease models. The recent discovery that certain transition metal carbonyls function as CO-releasing molecules (CO-RMs) in biological systems highlighted the potential of exploiting this and similar classes of compounds as a stratagem to deliver CO for therapeutic purposes. Here we describe the biochemical features and pharmacological actions of a newly identified water-soluble CO releaser (CORM-A1) that, unlike the first prototypic molecule recently described (CORM-3), does not contain a transition metal and liberates CO at a much slower rate under physiological conditions. Using a myoglobin assay and an amperometric CO electrode, we demonstrated that the release of CO from CORM-A1 is both pH- and temperature-dependent with a half-life of approximately 21 min at 37 degrees C and pH 7.4. In isolated aortic rings, CORM-A1 promoted a gradual but profound concentration-dependent vasorelaxation over time, which was highly amplified by YC-1 (1 microM) and attenuated by ODQ, a stimulator and inhibitor of guanylate cyclase, respectively. Similarly, administration of CORM-A1 (30 micromol/kg i.v.) in vivo produced a mild decrease in mean arterial pressure, which was markedly potentiated by pretreatment with YC-1 (1.2 micromol/kg i.v.). Interestingly, an inactive form of CORM-A1 that is incapable of releasing CO failed to promote both vasorelaxation and hypotension, thus directly implicating CO as the mediator of the observed pharmacological effects. Our results reveal that the bioactivities exerted by CORM-A1 reflect its intrinsic biochemical behavior of a slow CO releaser, which may be advantageous in the treatment of chronic conditions that require CO to be delivered in a carefully controlled manner.

Carbon monoxide-releasing molecules (CO-RMs) attenuate the inflammatory response elicited by lipopolysaccharide in RAW264.7 murine macrophages.

The enzyme heme oxygenase-1 (HO-1) is a cytoprotective and anti-inflammatory protein that degrades heme to produce biliverdin/bilirubin, ferrous iron and carbon monoxide (CO). The anti-inflammatory properties of HO-1 are related to inhibition of adhesion molecule expression and reduction of oxidative stress, while exogenous CO gas treatment decreases the production of inflammatory mediators such as cytokines and nitric oxide (NO). CO-releasing molecules (CO-RMs) are a novel group of substances identified by our group that are capable of modulating physiological functions via the liberation of CO. We aimed in this study to examine the potential anti-inflammatory characteristics of CORM-2 and CORM-3 in an in vitro model of lipopolysaccharide (LPS)-stimulated murine macrophages. Stimulation of RAW264.7 macrophages with LPS resulted in increased expression of inducible NO synthase (iNOS) and production of nitrite. CORM-2 or CORM-3 (10-100 microM) reduced nitrite generation in a concentration-dependent manner but did not affect the protein levels of iNOS. CORM-3 also decreased nitrite levels when added 3 or 6 h after LPS exposure. CORM-2 or CORM-3 did not cause any evident cytotoxicity and produced an increase in HO-1 expression and heme oxygenase activity; this effect was completely prevented by the thiol donor N-acetylcysteine. CORM-3 also considerably reduced the levels of tumor necrosis factor-alpha, another mediator of the inflammatory response. The inhibitory effects of CORM-2 and CORM-3 were not observed when the inactive compounds, which do not release CO, were coincubated with LPS. These results indicate that CO liberated by CORM-2 and CORM-3 significantly suppresses the inflammatory response elicited by LPS in cultured macrophages and suggest that CO carriers can be used as an effective strategy to modulate inflammation.

[Mn(CO)4{S2CNMe(CH2CO2H)}], a new water-soluble CO-releasing molecule.

[Mn(CO)(4){S(2)CNMe(CH(2)CO(2)H)}], 1, is shown to be a CO releasing molecule providing at least three moles CO per mole of compound. The mechanism of CO loss is dissociative and reversible and was investigated using Gaussian 09 calculations. The reversible binding of CO results in a relatively stable solution of the compound, while in the presence of a CO receptor or a ligand to prevent the rebinding of CO, the CO is lost rapidly. The X-ray structure was determined.

A re-investigation of [Fe(L-cysteinate)2(CO)2]2-: an example of non-heme CO coordination of possible relevance to CO binding to ion channel receptors.

[Fe(L-cysteinate)(2)(CO)(2)](2-) is a CO releasing molecule which has low cytotoxicity to RAW264.7 macrophages. It provides an example of CO binding using ligands available to ion channels which use CO as a signalling molecule in the absence of heme. Previous work has shown that this compound consists of five isomers and it was proposed that the two isomers with trans-dicarbonyls are dominant. In this work the isomers are re-assigned and shown to be capable of releasing CO, albeit too slowly to act as a signalling receptor. It is shown that by linking the two L-cysteines together to form [Fe(SCH(2)CH{CO(2)H}NHCH(2))(2)(CO)(2)], only one isomer is isolated.

Modification of the deoxy-myoglobin/carbonmonoxy-myoglobin UV-vis assay for reliable determination of CO-release rates from organometallic carbonyl complexes.

The deoxy-myoglobin (deoxy-Mb)/carbonmonoxy-myoglobin (Mb-CO) UV-vis assay is the principal method used for quantifying the rates of CO release from CO-releasing molecules (CO-RMs) that might possess therapeutic benefits. Some issues emerge when the Mb-CO assay is utilized for testing CO-RMs with novel structures, which are comprehensively discussed here for the first time. Two methods for processing raw UV-vis spectroscopic data generated from the assay are presented in this paper.

Iron indenyl carbonyl compounds: CO-releasing molecules.

New CO-releasing molecules, CO-RMs, based on indenyl iron carbonyls have been identified. Half-lives for carbon monoxide (CO) release, (1)H NMR, (13)C NMR, IR, mass spectra, elemental analysis and biological data have been determined for the compounds. Limited correlations have been made between half-lives and ΔG(‡) for CO release and spectroscopic parameters, ν(CO) and δ((13)CO). X-ray structures have been determined for [Fe(η(5)-C(9)H(7))(CO)(2)L][BF(4)] where L is CO, NCMe, PPh(3), P(OPh)(3), NC(5)H(5) or 1-methylimidazole. Improved preparations of [Fe(η(5)-C(9)H(7))(CO)(2)](2) and [Fe(η(5)-C(9)H(7))(CO)(3)][BF(4)] are reported.

Mu2-alkyne dicobalt(0)hexacarbonyl complexes as carbon monoxide-releasing molecules (CO-RMs): probing the release mechanism.

The first carbon monoxide-releasing molecules (CO-RMs) based on mu2-alkyne dicobalt(0)hexacarbonyl complexes are reported. The alkyne substituents significantly affect the rate of CO-release, cytotoxicity and cell viability. Mechanistic studies provide insight into the CO-RM activation pathways.

Donor HO-1 expression inhibits intimal hyperplasia in unmanipulated graft recipients: a potential role for CD8+ T-cell modulation by carbon monoxide.

BACKGROUND: Induction of heme oxygenase (HO)-1 expression protects transplanted organs from humoral rejection and ischemia-reperfusion injury, but induction in recipient immune cells also has direct immunomodulatory effects. Although many studies have examined the impact of HO-1 after transplantation, it is still unclear whether HO-1 expression solely in the donor tissue can influence the recipient T-cell response. METHODS: Donor mice were treated with hemin to transiently upregulate HO-1. Control or HO-1-expressing aortas were transplanted into fully mismatched, completely unmanipulated recipients, and harvested at 6 weeks to assess neointimal area and T-cell infiltration. T cells were isolated from draining lymph nodes to assess cytokine production. In vitro, T-cell proliferative and cytokine responses to allogeneic donor dendritic (DC) and endothelial cells expressing HO-1 were examined. RESULTS: Neointimal area was significantly (P<0.01) reduced in HO-1-expressing grafts. Hemin pretreated endothelial cells significantly inhibited proliferation (P<0.01) and interferon (IFN)-gamma production (P =0.01) in allogeneic CD8 T cells. This effect was mimicked by a carbon monoxide-releasing molecule. No phenotypic or functional changes were observed after incubation of T cells with hemin-treated dendritic cells. T-cell infiltration of HO-1-expressing donor aortas was significantly reduced (P<0.001), but proportions of IFN-gamma-producing T cells harvested from regional lymph nodes were similar. CONCLUSIONS: Organs expressing cytoprotective HO-1 have a direct influence on the recipient immune response. Given the important role of CD8 T cells and IFN-gamma in chronic rejection, these data suggest that donor HO-1 expression may be useful to augment other immunosuppressive therapies to prolong graft survival and inhibit intimal hyperplasia.

Eta(1)-2-pyrone metal carbonyl complexes as CO-releasing molecules (CO-RMs): a delicate balance between stability and CO liberation.

An evaluation of the CO releasing ability of iron(II) and molybdenum(II) complexes has facilitated the discovery of the most rapid CO releaser, namely [Mo(CO)(3)(eta(5)-C(5)H(5))(eta(1)-{O}-C{=O}-O-CMe=CH-COMe=CBr)]BF(4) (CORM-F10), reported to date. The rate of CO release is related to the overall solution phase stability of the transition metal carbonyl complex. The cytotoxicity and vasodilatory properties of CORM-F10 have been determined.

[(Eta-C5H4R)Fe(CO)2X], X = Cl, Br, I, NO3, CO2Me and [(eta-C5H4R)Fe(CO)3]+, R = (CH2)nCO2Me (n = 0-2), and CO2CH2CH2OH: a new group of CO-releasing molecules.

A new group of CO-releasing molecules, CO-RMs, based on cyclopentadienyl iron carbonyls have been identified. X-Ray structures have been determined for [(eta-C(5)H(4)CO(2)Me)Fe(CO)(2)X], X = Cl, Br, I, NO(3), CO(2)Me, [(eta-C(5)H(4)CO(2)Me)Fe(CO)(2)](2), [(eta-C(5)H(4)CO(2)CH(2)CH(2)OH)Fe(CO)(2)](2) and [(eta-C(5)H(4)CO(2)Me)Fe(CO)(3)][FeCl(4)]. Half-lives for CO release, (1)H, (13)C, and (17)OC NMR and IR spectra have been determined along with some biological data for these compounds, [(eta-C(5)H(4)CO(2)CH(2)CH(2)OH)Fe(CO)(3)](+) and [[eta-C(5)H(4)(CH(2))(n)CO(2)Me]Fe(CO)(3)](+), n = 1, 2. More specifically, cytotoxicity assays and inhibition of nitrite formation in stimulated RAW264.7 macrophages are reported for most of the compounds analyzed. [(eta-C(5)H(5))Fe(CO)(2)X], X = Cl, Br, I, were also examined for comparison. Correlations between the half-lives for CO release and spectroscopic parameters are found within each group of compounds, but not between the groups.

Eta4-pyrone iron(0)carbonyl complexes as effective CO-releasing molecules (CO-RMs).

The CO-releasing properties of iron(0)tricarbonyl complexes bearing a 2-pyrone ligand have been evaluated. In this report, we demonstrate that the intrinsic stability of the (eta4-2-pyrone)Fe(CO)3 complex influences the extent and rate of CO release, which is affected by the presence of a halogen substituent on the 2-pyrone ring. The cell viability index has been highlighted for the active carbon monoxide-releasing molecules (CO-RMs), demonstrating that these complexes and related derivatives are a promising new class of compounds with potential therapeutic applications.

Bioactive properties of iron-containing carbon monoxide-releasing molecules.

Carbon monoxide-releasing molecules (CO-RMs) are compounds capable of delivering controlled amounts of CO within a cellular environment. Ruthenium-based carbonyls [tricarbonyldichloro ruthenium(II) dimer and tricarbonylchloro-(glycinato)ruthenium(II)] and boronacorbonates (sodium boranocarbonate) have been shown to promote vasodilatory, cardioprotective, and anti-inflammatory activities in a variety of experimental models. Here, we extend our previous studies by showing that eta-4-(4-bromo-6-methyl-2-pyrone)tricarbonyl iron (0) (CORM-F3), an irontricarbonyl complex that contains a 2-pyrone motif, liberates CO in vitro and exerts pharmacological actions that are typical of CO gas. Specifically, CORM-F3 caused vasorelaxation in isolated aortic rings and inhibited the inflammatory response (e.g., nitrite production) of RAW264.7 macrophages stimulated with endotoxin in a dose-dependent fashion. By analyzing the rate of CO release, we found that when the bromide at the 4-position of the 2-pyrone CORM-F3 is substituted with a chloride group [eta-4-(4-chloro-6-methyl-2-pyrone)tricarbonyl iron (0) (CORM-F8)], the rate of CO release is significantly decreased (4.5-fold), and a further decrease is observed when the 4- and 6-positions are substituted with a methyl group [eta-4-(4-methyl-6-methyl-2-pyrone)tricarbonyl iron (0) (CORM-F11)] or a hydrogen [eta-4-(4-chloro-2-pyrone)tricarbonyl iron (0) (CORM-F7)], respectively. Interestingly, the compounds containing halogens at the 4-position and the methyl at the 6-position of the 2-pyrone ring (CORM-F3 and CORM-F8) were found to be less cytotoxic compared with other CO-RMs when tested in RAW246.7 macrophages. Thus, iron-based carbonyls mediate pharmacological responses that are achieved through liberation of CO and the nature of the substituents in the organic ligand have a profound effect on both the rate of CO release and cytotoxicity.