Legacy effect of short-term alirocumab in familial hypercholesterolaemia: A case report.

Homozygous familial hypercholesterolaemia (FH) is a rare genetic disorder with aberrantly high level of low-density lipoprotein cholesterol (LDL-C) requiring multiple combined aggressive lipidlowering therapy to reduce the progression of atherosclerotic cardiovascular disease. Alirocumab, a proprotein convertase subtilisin/kexin type 9 inhibitor (PCSK9i) has been approved for treatment of FH, which requires further lowering of LDL-C in addition to diet modification and maximally tolerated statin therapy. We report the response of short-term alirocumab treatment on a young patient with clinically and genetically confirmed FH, who suffered from acute coronary syndrome, and in particular, discussed the hypothesised legacy effect of PCSK9i. The patient was initially treated with a combination of high-intensity statin and ezetimibe for 12 weeks. Subsequently, alirocumab was added to the patient's lipid-lowering regime and he managed to attain guideline recommended LDL-C target within 10 weeks. However, alirocumab was stopped at week 54 due to financial constraint. Interestingly, despite cessation of PCSK9i therapy for a period of 30 weeks, the patient's LDL-C level rose slightly not returning to his baseline level.

A successful pregnancy outcome of homozygous familial hypercholesterolaemia patient on statin therapy.

Homozygous familial hypercholesterolaemia (HoFH) is a rare genetic disorder of lipoprotein metabolism mainly due to mutation of the low-density lipoprotein (LDL)-receptor gene (LDLR). It is a life-threatening disease that causes accelerated, multi-vessel atherosclerosis presented in early childhood. Pregnancy in HoFH may pose early coronary morbidity and mortality to both the foetus and mother. The combination of HoFH and pregnancy can be a fatal condition. While statins are very effective in lowering low-density lipoprotein cholesterol (LDL-C) levels, they are generally contraindicated during pregnancy, thus their use during pregnancy is uncommon. On the other hand, lipid apheresis (LA) has turned into an effective treatment to control cholesterol level amid pregnancy. However, the procedure is not widely available in our region. To date, there are scarcely documented case reports of HoFH in pregnancy in which the majority of them underwent LA to keep LDL-C at a low level. We report a rare case of successful pregnancy outcome of HoFH patient treated with lipid-lowering drugs including statin without LA therapy. Apart from that, we also discussed the genetic findings of the proband and all screened family members in which to the best of our knowledge, the first study using the whole-exome sequencing technique to identify the causative gene mutations for familial hypercholesterolaemia among the Malaysian population.

Association of coronary artery calcium score with calcification and degree of stenosis: An autopsy study.

INTRODUCTION: Coronary artery disease (CAD) is a known cause of major cardiovascular events and calcium score (CS) has been developed as a marker of coronary atherosclerosis. Yet, the relationship between post mortem computed tomography (PMCT) CS with histologically observed calcification and the severity of coronary artery stenosis has not been widely explored and is still unclear. This study aims to determine the association between coronary artery PMCT CS with histologically observed calcification and degree of stenosis of coronary arteries in post-mortem cases. MATERIALS & METHODS: This was a cross-sectional study involving 101 subjects recruited from the National Institute of Forensic Medicine (IPFN) Hospital Kuala Lumpur (HKL) over a period of 15 months, from December 2012 until April 2014. PMCT CS of the coronary arteries was calculated using Agatston-Janowitz score. Histological presence of calcification was observed and the degree of stenosis was calculated using an image analysis technique. RESULTS: PMCT CS increased with increasing severity of stenosis (p<0.001). PMCT CS showed a positive correlation with the presence of calcification (r=-0.82, p<0.001). CONCLUSION: Calcium score is strongly associated with coronary artery calcification and the degree of luminal stenosis in post mortem subjects. Thus, PMCT may be useful as a non-invasive tool in diagnosing CAD in the event that an autopsy is not possible.

Effects of prolonged isolation in a confined space on status of oxidative stress and prothrombogenesis: In preparation for possible future manned space expedition to Mars.

INTRODUCTION: Apart from inflammation and endothelial dysfunction, other key components in the development of atherogenesis include prothrombogenesis and oxidative stress. The effects of long-term confinement and isolation, exposure to radiation and different gravity forces during space travel could potentially increase the long-term risk of atherosclerosis. To the best of our knowledge, this is the first study determining the status of prothrombogenesis and oxidative stress in six cosmonauts subjected to the longest duration of confined isolation period of 520 days in preparation for prospective undetermined manned space travel to Mars. MATERIALS AND METHODS: This collaborative research between the National Space Agency (ANGKASA), Universiti Teknologi MARA, Malaysia and Institute of Biomedical Problems (IBMP), Russia was conducted at the Russian Academy of Sciences IBMP, Moscow, Russia. Six multi-national cosmonauts were assigned to live in a ground-based confined module for 520 days. Standard exercise and diet regime were instituted throughout the isolation phase. Six age, ethnic and gender-matched healthy, free-living ground controls were recruited in parallel. Serial serum and whole blood were analysed for biomarkers of prothrombogenesis [plasminogen activator inhibitor-1 (PAI-1) and homocysteine] and oxidative stress [oxidised low-density lipoprotein (ox-LDL) and malondialdehyde (MDA)]. RESULTS: There were significantly lower concentrations of PAI-1 and homocysteine in cosmonauts during confinement compared to the controls. There were no significant differences seen in the concentrations of biomarkers of oxidative stress during confinement but there was a significant percentage change increment for serum MDA in cosmonauts. CONCLUSION: Long-term confinement decreased the risk of prothrombogenesis and this could be attributed to the exercise and diet regime which includes omega-3 fatty acids supplementation given to the crew members during their confinement period. However, oxidative damage could not be excluded and may be attributed to the influence of psychological stress during this prolonged confinement.

Sudden death due to infective endocarditis caused by Streptococcus constellatus: A case report.

Infective endocarditis (IE) is a relatively uncommon disease, but has been challenging to diagnose over the years. With the increasing incidence, variety of causative agents and the resistance of microorganisms towards antibiotics, there is still an occurrence of sudden death due to undiagnosed IE. The most common microorganism causing IE is Staphylococcus aureus. However, there is increasing prevalence of other microorganisms causing IE. This case report highlights a case of sudden death due to IE caused by a rare pathogen, Streptococcus constellatus which belongs to the Streptococcus anginosus group (Milleri group). A study noted the crude incidence of IE in 6 world regions ranged between 1.5 and 11.6 cases per 100,000 people. To date, there has been no previous report on sudden death due to IE caused by Streptococcus constellatus in Malaysia, neither in the forensic nor clinical setting. This case report underlined the characteristics and pathological features of this microorganism. The increasing incidence and variety of causative organisms in IE are important public health issues. It is vital for future studies to examine the risk factors of IE related to Streptococcus constellatus, to enhance better understanding, insight and awareness regarding the course of this disease. This in turn may facilitate preventive measures to avoid morbidity and mortality from this condition.

Updates in the management of Dyslipidaemia in the high and very high risk individual for CV risk reduction.

Cardiovascular disease (CVD) has been the main cause of mortality and an important cause of morbidity in Malaysia for several years. To reduce global cardiovascular (CV) risk in the population, primary preventive strategies need to be implemented. Hypercholesterolaemia is one of the major risk factors for CVD. This paper is an expert review on the management of hypercholesterolemia focusing on high and very high risk individuals. In low and Intermediate risk individuals, therapeutic lifestyle changes (TLC) and a healthy lifestyle alone may suffice. In high and very high risk individuals, drug therapy in conjunction with TLC are necessary to achieve the target LDL-C levels which have been shown to slow down progression and sometimes even result in regression of atherosclerotic plaques. Statins are first-line drugs because they have been shown in numerous randomized controlled trials to be effective in reducing CV events and to be safe. In some high risk individuals, despite maximally tolerated statin therapy, target Low Density Lipoprotein Cholesterol (LDL-C) levels are not achieved. These include those with familial hypercholesterolaemia and statin intolerance. This paper discusses non-statin therapies, such as ezetimibe and the newer Proprotein convertase subtilisin/kexin type 9 Inhibitors (PCSK9-i).

Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro.

Prolonged disuse of the musculoskeletal system is associated with reduced mechanical loading and lack of anabolic stimulus. As a form of mechanical signal, the multidirectional orbital fluid shear stress transmits anabolic signal to bone forming cells in promoting cell differentiation, metabolism and proliferation. Signals are channeled through the cytoskeleton framework, directly modifying gene and protein expression. For that reason, we aimed to study the organization of Normal Human Osteoblast (NHOst) cytoskeleton with regards to orbital fluid shear (OFS) stress. Of special interest were the consequences of cytoskeletal reorganization on NHOst metabolism, proliferation, and osteogenic functional markers. Cells stimulated at 250 RPM in a shaking incubator resulted in the rearrangement of actin and tubulin fibers after 72 h. Orbital shear stress increased NHOst mitochondrial metabolism and proliferation, simultaneously preventing apoptosis. The ratio of RANKL/OPG was reduced, suggesting that orbital shear stress has the potential to inhibit osteoclastogenesis and osteoclast activity. Increase in ALP activity and OCN protein production suggests that stimulation retained osteoblast function. Shear stress possibly generated through actin seemed to hold an anabolic response as osteoblast metabolism and functional markers were enhanced. We hypothesize that by applying orbital shear stress with suitable magnitude and duration as a non-drug anabolic treatment can help improve bone regeneration in prolonged disuse cases.

Homozygous familial hypercholesterolemia.

We report a rare case of homozygous familial hypercholesterolemia (HoFH), a 22-year-old Malay woman who presented initially with minor soft tissue injury due to a cycling accident. She was then incidentally found to have severe xanthelasma and hypercholesterolemia (serum TC 15.3 mmol/L and LDL-C 13.9 mmol/L). She was referred to the Specialized Lipid Clinic and was diagnosed with familial hypercholesterolemia (FH) based on the Simon Broome (SB) diagnostic criteria. There was a family history of premature coronary heart disease (CHD) in that three siblings had sudden cardiac death, and of consanguineous marriage in that her parents are cousins. DNA screening of LDLR and APOB genes was done by Polymerase Chain Reaction (PCR), followed by Denaturing High Performance Liquid Chromatography (DHPLC). Homozygous mutation C255S in Exon 5 of her LDLR gene was found. There was no mutation was found in Exon 26 and Exon 29 of the APOB gene. This report is to emphasize the importance of identifying patients with FH and cascade screening through established diagnostic criteria and genetic studies in order to ensure early detection and early treatment intervention to minimize the risk of developing CHD and related complications.

Short-term moderate hypothermia stimulates alkaline phosphatase activity and osteocalcin expression in osteoblasts by upregulating Runx2 and osterix in vitro.

Exposure of Normal Human Osteoblast cells (NHOst) to a period of hypothermia may interrupt their cellular functions, lead to changes in bone matrix and disrupt the balance between bone formation and resorption, resulting in bone loss or delayed fracture healing. To investigate this possibility, we exposed NHOst cells to moderate (35 °C) and severe (27 °C) hypothermia for 1, 12, 24 and 72 h. The effects of hypothermia with respect to cell cytoskeleton organization, metabolic activity and the expression of cold shock chaperone proteins, osteoblast transcription factors and functional markers, were examined. Our findings showed that prolonged moderate hypothermia retained the polymerization of the cytoskeletal components. NHOst cell metabolism was affected differently according to hypothermia severity. The osteoblast transcription factors Runx2 and osterix were necessary for the transcription and translation of bone matrix proteins, where alkaline phosphatase (Alp) activity and osteocalcin (OCN) bone protein were over expressed under hypothermic conditions. Consequently, bone mineralization was stimulated after exposure to moderate hypothermia for 1 week, indicating bone function was not impaired. The cold shock chaperone protein Rbm3 was significantly upregulated (p<0.001) during the cellular stress adaption under hypothermic conditions. We suggest that Rbm3 has a dual function: one as a chaperone protein that stabilizes mRNA transcripts and a second one in enhancing the transcription of Alp and Ocn genes. Our studies demonstrated that hypothermia permitted the in vitro maturation of NHOst cells probably through an osterix-dependent pathway. For that reason, we suggest that moderate hypothermia can be clinically applied to counteract heat production at the fracture site that delays fracture healing.

Morphological and phenotypical characteristics of human osteoblasts after short-term space mission.

Morphological and phenotypical signs of cultured readaptation osteoblasts were studied after a short-term space mission. The ultrastructure and phenotype of human osteoblasts after Soyuz TMA-11 space flight (2007) were evaluated by scanning electron microscopy, laser confocal microscopy, and ELISA. The morphofunctional changes in cell cultures persisted after 12 passages. Osteoblasts retained the drastic changes in their shape and size, contour deformation, disorganization of the microtubular network, redistribution of organelles and specialized structures of the plasmalemma in comparison with the ground control cells. On the other hand, the expression of osteoprotegerin and osteocalcin (bone metabolism markers) increased; the expression of bone resorption markers ICAM-1 and IL-6 also increased, while the expression of VCAM-1 decreased. Hence, space flight led to the development of persistent shifts in cultured osteoblasts indicating injuries to the cytoskeleton and the phenotype changes, indicating modulation of bone metabolism biomarkers.

Alteration of cell cytoskeleton and functions of cell recovery of normal human osteoblast cells caused by factors associated with real space flight.

Experiments involving short-term space flight have shown an adverse effect on the physiology, morphology and functions of cells investigated. The causes for this effect on cells are: microgravity, temperature fluctuations, mechanical stress, hypergravity, nutrient restriction and others. However, the extent to which these adverse effects can be repaired by short-term space flown cells when recultured in conditions of normal gravity remains unclear. Therefore this study aimed to investigate the effect of short-term spaceflight on cytoskeleton distribution and recovery of cell functions of normal human osteoblast cells. The ultrastructure was evaluated using ESEM. Fluorescent staining was done using Hoechst, Mito Tracker CMXRos and Tubulin Tracker Green for cytoskeleton. Gene expression of cell functions was quantified using qPCR. As a result, recovered cells did not show any apoptotic markers when compared with control. Tubulin volume density (p<0.001) was decreased significantly when compared to control, while mitochondria volume density was insignificantly elevated. Gene expression for IL-6 (p<0.05) and sVCAM-1 (p<0.001) was significantly decreased while alkaline phosphatase (p<0.001), osteocalcin and sICAM (p<0.05) were significantly increased in the recovered cells compared to the control ones. The changes in gene and protein expression of collagen 1A, osteonectin, osteoprotegerin and beta-actin, caused by short-term spaceflight, were statistically not significant. These data indicate that short term space flight causes morphological changes in osteoblast cells which are consistent with hypertrophy, reduced cell differentiation and increased release of monocyte attracting proteins. The long-term effect of these changes on bone density and remodeling requires more detailed studies.

Interleukin-6 and intercellular cell adhesion molecule-1 expression remains elevated in revived live endothelial cells following spaceflight.

The effects of spaceflight on cardiovascular health are not necessarily seen immediately after astronauts have returned but can be delayed. It is important to investigate the long term effects of spaceflight on protein and gene expression of inflammation and endothelial activation as a predictor for the development of atherosclerosis and potential cardiovascular problems. The objectives of this study were to investigate the (a) protein and gene expression of inflammation and endothelial activation, (b) expression of nuclear factor kappa B (NFκB), signal transducer and activator of transcription-3 (STAT-3) and endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVEC) 3 months post-space flight travel compared to ground controls. HUVEC cultured on microcarriers in fluid processing apparatus were flown to the International Space Station (ISS) by the Soyuz TMA-11 rocket. After landing, the cells were detached from microcarriers and recultured in T-25 cm(2) culture flasks (Revived HUVEC). Soluble protein expression of IL-6, TNF-α, ICAM-1, VCAM-1 and e-selectin were measured by ELISA. Gene expression of these markers and in addition NFκB, STAT-3 and eNOS were measured. Spaceflight induced IL-6 and ICAM-1 remain elevated even after 3 months post spaceflight travel and this is mediated via STAT-3 pathway. The downregulation of eNOS expression in revived HUVEC cells suggests a reduced protection of the cells and the surrounding vessels against future insults that may lead to atherosclerosis. It would be crucial to explore preventive measures, in relation to atherosclerosis and its related complications.

Effects of space mission factors on the morphology and function of endothelial cells.

The structure and functions of endothelial cells after space mission were studied by electron and laser confocal microscopy, image analysis, and MTT test. The endothelial cells changed significantly (proliferative activity, size, contours, shape, distribution of mitochondria and microtubules) in comparison with controls on the Earth. These changes indicated injuries in the cytoskeleton and impairment of the barrier function of the cells, which presumably contributed to the development of endothelial dysfunction.

Real space flight travel is associated with ultrastructural changes, cytoskeletal disruption and premature senescence of HUVEC.

Microgravity, hypergravity, vibration, ionizing radiation and temperature fluctuations are major factors of outer space flight affecting human organs and tissues. There are several reports on the effect of space flight on different human cell types of mesenchymal origin while information regarding changes to vascular endothelial cells is scarce. Ultrastructural and cytophysiological features of macrovascular endothelial cells in outer space flight and their persistence during subsequent culturing were demonstrated in the present investigation. At the end of the space flight, endothelial cells displayed profound changes indicating cytoskeletal lesions and increased cell membrane permeability. Readapted cells of subsequent passages exhibited persisting cytoskeletal changes, decreased metabolism and cell growth indicating cellular senescence.

In-vitro expression of adhesion molecules and bone specific markers are depending on the cell culture material.

Bone formation is an active process whereby osteoblasts are found on the surface of the newly formed bone. Adhesion to extracellular matrix is essential for the development of bone however not all surfaces are suitable for osteoblast adhesion and don't support osteoblastic functions. The objective of this study was to test the suitability of a collagen based microcarrier which would support osteoblastic functions.

The prognostic value of C-reactive protein (CRP) levels in patients with acute ischaemic stroke.

Increasing evidence suggests that inflammation plays an important role in the development of both cardiovascular and cerebrovascular events. Recently C-reactive protein (CRP) levels have been reported to be a prognostic factor for cerebrovascular and cardiovascular events. The main objective of the study was to evaluate the prognostic value of CRP levels in a first ever ischaemic stroke at one month. All ischaemic stroke patients who were admitted to Hospital Universiti Kebangsaan Malaysia (HUKM) between May 2002 and July 2002 were eligible for the study. CRP levels were taken within 72 hours after an acute ischaemic stroke. The functional ability was assessed using the Barthel Index (BI) after one month of stroke. During the study period 84 patients were admitted to HUKM with the diagnosis of ischaemic stroke; 49 patients were enrolled and 35 were excluded. Twenty-nine patients (59.2%) had elevated CRP levels (median 1.64+/-3.07 mg/dL, range 0.06 to 16.21 mg/dL). Elevated CRP levels were found to be a predictor of severe functional disability (BI<5) and were also associated with larger infarcts. In conclusion, elevated CRP levels are associated with poorer functional outcome and predict a larger infarct size.

Reduction in serum levels of adhesion molecules, interleukin-6 and C-reactive protein following short-term low-dose atorvastatin treatment in patients with non-familial hypercholesterolemia.

Hypercholesterolemia causes endothelial dysfunction, an early feature of atherosclerosis, leading to increased production of adhesion molecules and cytokines. The aim of this study was to investigate the effects of three months of treatment with low dose atorvastatin on serum levels of adhesion molecules, interleukin-6 (IL-6) and highly sensitive C-reactive protein (hs-CRP) in patients with non-familial hypercholesterolemia. Fifty-five patients with non-familial hypercholesterolemia were randomized to treatment with atorvastatin 10 mg/day or placebo for 3 months. Soluble intercellular adhesion molecules-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, IL-6 and hs-CRP levels were measured to assess the inflammatory activity of the endothelium. There was a significant reduction in ICAM-1 at 2 weeks (p<0.0001) with further reduction at 3 months (p<0.0001). At 3 months, there were significant reductions in VCAM-1 (p<0.02), IL-6 (p<0.0001) and hs-CRP (p<0.01), but an increase in E-selectin levels (p<0.002). Treatment with statin was an independent determinant of change in ICAM-1 (p<0.05) and IL-6 levels (p<0.05) after correcting for anthropometric indices, blood pressure and lipid profile. Low-dose atorvastatin treatment leads to reduction in proinflammatory markers of endothelial function, suggesting an attenuation of endothelial activation and improvement in endothelial function, independent of lipid lowering. This may lead to a reduction in the progression of atherosclerosis.

Soluble intercellular adhesion molecule-1 and interleukin-6 levels reflect endothelial dysfunction in patients with primary hypercholesterolaemia treated with atorvastatin.

Adhesion molecules and cytokines are involved in the pathogenesis of intimal injury in atherosclerosis but their relationship with endothelial function remains unclear. The objectives of this study were to examine the effects of atorvastatin on soluble adhesion molecules, interleukin-6 (IL-6) and brachial artery endothelial-dependent flow mediated dilatation (FMD) in patients with familial (FH) and non-familial hypercholesterolaemia (NFH). A total of 74 patients (27 FH and 47 NFH) were recruited. Fasting lipid profiles, soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular-cellular adhesion molecule-1 (sVCAM-1), E-selectin, IL-6 and FMD were measured at baseline, 2 weeks, 3 and 9 months post-atorvastatin treatment (FH--80 mg/day, NFH--10 mg/day). In both groups, compared to baseline, sICAM-1 levels were significantly reduced at 2 weeks, further reduced at 3 months and maintained at 9 months (P<0.0001). The IL-6 levels were significantly reduced at 3 months and 9 months compared to baseline for FH (P<0.005) and NFH (P<0.0001). In both groups, the FMD at 2 weeks was higher than baseline (P<0.005), with progressive improvement up to 9 months. FMD was negatively correlated with sICAM-1 and IL-6. In conclusion, both low and high doses of atorvastatin lead to early progressive improvement in endothelial function in patients with primary hypercholesterolaemia. sICAM-1 and IL-6 levels reflect endothelial dysfunction in these patients.

Clinical usefulness of tumour markers.

Tumour markers are substances related to the presence or progress of a tumour. An ideal tumour marker is (1) detectable only when malignancy is present, (2) specific for the type and site of malignancy, (3) correlates with the amount of malignant tissue present and (4) responds rapidly to a change in tumour size. At present, no tumour marker fulfills all of the above criteria. The first part of the review discusses the clinical usefulness of the commonly requested serum tumour markers, namely, prostate-specific antigen (PSA), CA 19-9, carcinoembryonic antigen (CEA), CA 125, CA 15-3, human chorionic gonadotrophin (hCG) and alpha-foetoprotein (AFP). It is hoped that this review article will decrease the abuse and misuse of these commonly requested serum tumour markers. The second part of the review discusses the clinical usefulness of catecholamines and their metabolites, calcitonin, thyroglobulin, parathyroid hormone, prolactin, adrenocorticotrophic hormone, oestrogen and progesterone receptors, p53, HER-2/c-erbB2, BRCA1 and BRCA2.

Measurement of intima-media thickness of common carotid arteries using ultrasound in patients with familial and non-familial hypercholesterolaemia and correlation of intima-media thickness to obesity.

Ultrasonographic measurements of the intima-media thickness (IMT) of common carotid arteries (CCA) were taken in 50 patients with familial hypercholesterolaemia (FH) and 57 patients with non-familial hypercholesterolemia (NFH). The lipid profile, body mass index (BMI) and waist-hip ratio (WHR) of each patient were recorded. In FH patients, the IMT was significantly higher in overweight and elevated WHR subgroups compared to the normal with significant correlations between BMI and WHR to the IMT. In NFH patients, the IMT was significantly higher in the elevated WHR compared to the normal subgroup but the correlations between either BMI or WHR to IMT were insignificant. These suggest that the environmentally modified anthropometric indices may have an effect on atherosclerosis in genetically determined hypercholesterolaemia in FH patients.