Andreas C. Gidlöf

Increased expression of inducible nitric oxide synthase in psoriatic skin and cytokine-stimulated cultured keratinocytes

Summary Since nitric oxide (NO) has been implicated in the pathogenesis of various hyperproliferative and inflammatory diseases, the mRNA expression of constitutive nitric oxide synthase (cNOS) and inducibie nitric oxide synthase (iNOS) were investigated in psoriatic skin by reverse transcriptase coupled to the polymerase chain reaction (PCR). The study showed that the mRNA expression of brain nitric oxide synthase (bNOS), one of two isoforms of cNOS, was weak in both psoriatic plaques lesions and uninvolved skin, while mRNA transcripts for the second isoform, endothelial nitric oxide synthase (eNOS). were not detectable using the present method. In contrast, the mRNA expression of iNOS was markedly increased in lesional skin as compared to uninvolved skin. Cultured human keratinocytes exposed to a combination of interleukin‐lβ (IL‐1β) and tumour necrosis factor‐α (TNF‐α) for 4 h. showed strong gene expression of iNOS, while in 24h. the expression had returned to baseline expression. In summary, the study demonstrates that mRNA for the inducible form of NOS is over‐expressed in psoriatic lesions. The cause of this may be the local presence of inflammatory cytokines. These findings imply that iNOS may play an important part in local regulation of NO synthesis in psoriasis and other inflammatory dermatoses.

Retinoic Acid Regulates Arterial Smooth Muscle Cell Proliferation and Phenotypic Features In Vivo and In Vitro Through an RARα-Dependent Signaling Pathway

We have recently shown that all-trans retinoic acid (tRA) modulates arterial smooth muscle cell (SMC) morphologic features and biochemical composition in vitro. It has been proposed that different SMC phenotypes coexist in arteries, which may be retrieved in culture: hence, a differential action of tRA on distinct SMC subsets is conceivable. We have examined the effect of tRA on SMC proliferation, migration, plasminogen activator activity, and alpha-smooth muscle actin expression in 2 phenotypically different rat SMC populations, cultured respectively from the normal aortic media and from the intimal thickening (IT) after endothelial injury. tRA inhibited proliferation and increased migration and tissue-type plasminogen activator activity in both SMC populations, but decreased alpha-smooth muscle actin only in SMC cultured from the IT. The action of tRA is mediated by 2 families of nuclear receptors, RAR and RXR, each containing 3 isoforms, alpha, beta, and gamma. RAR and RAR-alpha agonists, but not RXR agonists, inhibited SMC proliferation in both cell populations and alpha-smooth muscle actin expression only in IT SMC. When administered intraperitoneally to balloon-injured rats, tRA and RAR-alpha agonists reduced the intimal hyperplasia in the carotid artery. Our results show that tRA and synthetic retinoids can affect the proliferation, migration, and differentiation of SMC in vitro. Furthermore, retinoids are able to reduce the IT induced by endothelial injury in vivo.

CYP26B1 plays a major role in the regulation of all-trans-retinoic acid metabolism and signaling in human aortic smooth muscle cells

Aim: The cytochrome P450 enzymes of the CYP26 family are involved in the catabolism of the biologically active retinoid all-trans-retinoic acid (atRA). Since it is possible that an increased local CYP26 activity would reduce the effects of retinoids in vascular injury, we investigated the role of CYP26 in the regulation of atRA levels in human aortic smooth muscle cells (AOSMCs). Methods: The expression of CYP26 was investigated in cultured AOSMCs using real-time PCR. The metabolism of atRA was analyzed by high-performance liquid chromatography, and the inhibitor R115866 or small interfering RNA (siRNA) was used to suppress CYP26 activity/expression. Results: AOSMCs expressed CYP26B1 constitutively and atRA exposure augmented CYP26B1 mRNA levels. Silencing of the CYP26B1 gene expression or reduction of CYP26B1 enzymatic activity by using siRNA or the inhibitor R115866, respectively, increased atRA-mediated signaling and resulted in decreased cell proliferation. The CYP26 inhibitor also induced expression of atRA-responsive genes. Therefore, atRA-induced CYP26 expression accelerated atRA inactivation in AOSMCs, giving rise to an atRA-CYP26 feedback loop. Inhibition of this loop with a CYP26 inhibitor increased retinoid signaling. Conclusion: The results suggest that CYP26 inhibitors may be a therapeutic alternative to exogenous retinoid administration.

CYP26 Inhibitor R115866 Increases Retinoid Signaling in Intimal Smooth Muscle Cells

Objective—Intimal smooth muscle cells (SMCs) are dedifferentiated SMCs that have a powerful ability to proliferate and migrate. This cell-type is responsible for the development of intimal hyperplasia after vascular angioplasty. Retinoids, especially all-trans retinoid acid, are known to regulate many processes activated at sites of vascular injury, including modulation of SMC phenotype and inhibition of SMC proliferation. Intracellular levels of active retinoids are under firm control. A key enzyme is the all-trans retinoic acid-degrading enzyme cytochrome p450 isoform 26 (CYP26). Thus, an alternative approach to exogenous retinoid administration could be to increase the intracellular level of all-trans retinoic acid by blocking CYP26-mediated degradation of retinoids. Methods and Results—Vascular intimal and medial SMCs expressed CYP26A1 and B1 mRNA. Although medial cells remained unaffected, treatment with the CYP26-inhibitor R115866 significantly increased cellular levels of all-trans retinoic acid in intimal SMCs. The increased levels of all-trans retinoic acid induced retinoid-regulated genes and decreased mitogenesis. Conclusions—Blocking of the CYP26-mediated catabolism mimics the effects of exogenously administrated active retinoids on intimal SMCs. Therefore, CYP26-inibitors offer a potential new therapeutic approach to vascular proliferative disorders.

Differences in Retinol Metabolism and Proliferative Response between Neointimal and Medial Smooth Muscle Cells

Vascular disease is multifactorial and smooth muscle cells (SMCs) play a key role. Retinoids have been shown to influence many disease-promoting processes including proliferation and differentiation in the vessel wall. Phenotypic heterogeneity of vascular SMCs is a well-known phenomenon and phenotypic modulation of SMCs precedes intimal hyperplasia. The SMCs that constitute the intimal hyperplasia demonstrate a distinct phenotype and differ in gene expression compared to medial SMCs. Cellular retinol-binding protein-1 (CRBP-I), involved in retinoid metabolism, is highly expressed in intimal SMCs, indicating altered retinoid metabolism in this subset of cells. The aim of this study was to evaluate the metabolism of all-trans ROH (atROH), the circulating prohormone to active retinoids, in vascular SMCs of different phenotypes. The results show an increased uptake of atROH in intimal SMCs compared to medial SMCs as well as increased expression of the retinoid-metabolizing enzymes retinol dehydrogenase-5 and retinal dehydrogenase-1 and, in conjunction with this gene expression, increased production of all-trans retinoic acid (atRA). Furthermore, the retinoic acid-catabolizing enzyme CYP26A1 is expressed at higher levels in medial SMCs compared to intimal SMCs. Thus, both retinoid activation and deactivation processes are in operation. To analyze if the difference in ROH metabolism was also correlated to differences in the biological response to retinol, the effects of ROH on proliferation of SMCs with this phenotypic heterogeneity were studied. We found that intimal SMCs showed a dose- and time-dependent growth inhibition when treated with atROH in contrast to medial SMCs, in which atROH had a mitogenic effect. This study shows, for the first time, that (1) vascular SMCs are able to synthesize biologically active atRA from the prohormone atROH, (2) intimal SMCs have a higher capacity to internalize atROH and metabolize atROH into atRA compared to medial SMCs and (3) atROH inhibits growth of intimal SMCs, but induces medial SMC growth.

A CYP26B1 Polymorphism Enhances Retinoic Acid Catabolism and May Aggravate Atherosclerosis

All-trans retinoic acid, controlled by cytochrome P450, family 26 (CYP26) enzymes, potentially has beneficial effects in atherosclerosis treatment. This study investigates CYP26 subfamily B, polypeptide 1 (CYP26B1) in atherosclerosis and the effects of a genetic polymorphism in CYP26B1 on retinoid catabolism. We found that CYP26B1 mRNA was induced by retinoic acid in human atherosclerotic arteries, and CYP26B1 and the macrophage marker CD68 were colocalized in human atherosclerotic lesions. In mice, Cyp26B1 mRNA was higher in atherosclerotic arteries than in normal arteries. Databases were queried for nonsynonymous CYP26B1 single nucleotide polymorphisms (SNPs) and rs2241057 selected for further studies. Constructs of the CYP26B1 variants were created and used for production of purified proteins and transfection of macrophagelike cells. The minor variant catabolized retinoic acid with significantly higher efficiency, indicating that rs2241057 is functional and suggesting reduced retinoid availability in tissues with the minor variant. rs2241057 was investigated in a Stockholm Coronary Atherosclerosis Risk Factor (SCARF) subgroup. The minor allele was associated with slightly larger lesions, as determined by angiography. In summary, this study identifies the first CYP26B1 polymorphism that alters CYP26B1 capacity to metabolize retinoic acid. CYP26B1 was expressed in macrophage-rich areas of human atherosclerotic lesions, induced by retinoic acid and increased in murine atherosclerosis. Taken together, the results indicate that CYP26B1 capacity is genetically regulated and suggest that local CYP26B1 activity may influence atherosclerosis.

Vitamin A: a drug for prevention of restenosis/reocclusion after percutaneous coronary intervention?

The re-establishment of adequate blood flow in a vessel with a reduced lumen due to an atherosclerotic plaque by percutaneous vascular intervention is a well established procedure. However, the long-term outcome of such interventions is negatively influenced by the development of intimal hyperplasia/restenosis. Although extensively researched, this still represents a significant clinical problem. Retinoids, i.e. natural and synthetic derivates of vitamin A, represent a potential therapeutic compound, since they have been shown to influence the vast majority of processes that ultimately lead to reocclusion of the injured vessel. Retinoids exert their effects at the transcriptional level through their nuclear receptors. Targeting multiple processes, i.e. proliferation, migration, extracellular matrix composition and cell differentiation, as well as coagulation/fibrinolysis, should increase their future role in the prevention of restenosis. The purpose of this review is to summarize the diverse effects of retinoids on pathobiological and biological processes activated at sites of vascular injury with particular emphasis on intimal hyperplasia/restenosis after endovascular interventions.

Synthetic Retinoids Improve Survival in Rodent Model of Endotoxic Shock

OBJECTIVE To investigate the effect of synthetic retinoids on septic shock induced by lipopolysaccharide (LPS) in rats. DESIGN Randomised study. SETTINGS University hospital laboratory, Sweden. ANIMALS AND INTERVENTIONS 31 male Sprague Dawley rats randomised into four groups: controls, given vehicle alone (n = 6), LPS 6 mg/kg body weight alone (n = 12), and LPS 6 mg/kg but pretreated with the retinoic acid receptor-alpha (RAR-alpha) agonists CD336 (n = 6) and CD2081 (n = 7). MAIN OUTCOME MEASURES Arterial blood pressure and heart rate measured hourly for four hours; mortality. RESULTS LPS caused a pronounced fall in blood pressure within one hour of injection in all groups of rats. Of the 12 rats given LPS but not RAR-alpha agonists, 6 died before the end of the experiment. By contrast, all animal given either CD336 or CD2081 survived. The significantly improved survival was found despite no significant improvements in either mean arterial pressure or heart rate. CONCLUSION Pretreatment with selective synthetic RAR-alpha agonists improves survival after LPS-induced septic shock in rats. These agents may have therapeutic potential in the treatment of septic shock in humans.

Thioredoxin a novel biomarker of post-injury sepsis.

Background: Thioredoxin (TRX), an endogenous anti‐oxidant protein induced in inflammatory conditions, has been shown to increase in plasma and to be associated with outcome in septic patients. This biomarker has never been studied in a trauma setting. We hypothesized that TRX would be increased after trauma and associated with post‐injury sepsis. Methods: Single‐centre prospective observational study conducted at the intensive care unit (ICU) at the Karolinska University Hospital, Stockholm, Sweden, a level‐1 trauma centre. Eighty‐three severely injured trauma patients, 18 years or older, with an ICU stay of three days or more were included. Plasma samples were obtained on day 1 and 3 after informed consent. Clinical, physiological and outcome data were retrieved from the trauma and ICU research registries. Plasma samples were also obtained from 15 healthy subjects. In addition, a standardized porcine trauma model was conducted where a femur fracture followed by a controlled hemorrhage period were inflicted in four pigs. Results: In pigs, however not significant, there was a continuing increase in plasma‐TRX after femur fracture and sequential hemorrhage despite near normalisation of cardiac index and lactate levels. In patients, median injury severity score was 29 and 48 patients developed sepsis during their ICU stay. A three‐fold increase in initial TRX was seen in trauma patients when compared to healthy volunteers. Thioredoxin was significantly higher in patients in shock on admission, those subject to massive transfusion and in the most severely injured patients. No difference was seen between survivors and non‐survivors. Plasma‐TRX on day 1 was significantly increased in patients who later developed post‐injury sepsis. In a logistic regression analysis including TRX, C‐reactive protein, injury severity, massive transfusion, and admission blood pressure, TRX was the only variable independently associated with post‐injury sepsis. Conclusions: This study demonstrates that TRX is released into plasma in response to severe trauma and independently associated with post‐injury sepsis. The use of TRX as a biomarker in trauma patients needs further evaluation in larger studies. Level of evidence: Retrospective cohort study, level III. HIGHLIGHTSThioredoxin (TRX) in plasma is shown to be increased after traumatic injury.TRX levels correspond to injury severity, shock on arrival and massive transfusion.TRX is independently associated with later sepsis development.TRX shows promise as an early future biomarker for post‐injury sepsis.

Thioredoxin in Post-Injury Sepsis And Ards

Trauma is one of the leading causes of mortality worldwide, resulting in a great global burden of disability and mortality [1, 2]. Approximately half of all trauma deaths occur during the first hours due to traumatic brain injury or massive bleeding. Later deaths are due to, for example, sepsis or multiple organ failure [3]. Independent risk factors for post-injury sepsis are still somewhat poorly defined. Injury Severity Score (ISS), male gender, age, low Glasgow Coma Scale (GCS) and massive blood transfusion have been suggested as risk factors [4]. Thioredoxin (TRX) is thought to have important anti-oxidant properties but it also functions as an endogenous anti-inflammatory mediator [5]. The link between high plasma levels of TRX and sepsis has been studied previously, although with conflicting evidence [5, 6]. TRX-levels in trauma patients and the possible correlation to secondary complications such as post-injury sepsis and acute respiratory distress syndrome (ARDS) have to our knowledge not been studied previously.