Possible Biomarker for COX-2 Cardiovascular Risk

Die on Checkerboard
Image: Flickr - Cristian N Mormoloc
Selective COX-2 inhibitors were developed to minimise the adverse gastrointestinal effects seen with conventional NSAIDs and have provided effective pain relief for millions of arthritis patients. Long-term, high dosage use of some COX-2 inhibitors, however, was found to be associated with an increased risk of heart attacks and strokes, resulting in drug withdrawals. A clearer understanding of the mechanisms underlying the cardiovascular effects associated with COX-2 inhibitors would allow better risk/benefit assessment and could possibly lead to the development of safer inhibitors.

Researchers from the University of California, Davis and Beijing University have now shown that, in mice, oral administration of rofecoxib for 3 months leads to a more than 120-fold increase in the regulatory lipid, 20-hydroxyeicosatetraenoic acid (20-HETE) which correlates with a significantly shorter tail bleeding time. Further studies suggested that inhibition of COX-2-mediated 20-HETE degradation by rofecoxib may, at least in part, explain the increase in blood levels and shortened bleeding time and may also contribute to the cardiovascular side effects seen with rofecoxib. Although the relative importance of COX-2 in the metabolism of 20-HETE in man has not yet been determined, if it proves to be as important as in mice, blood levels of 20-HETE may be a good predictor of which patients are at higher risk of heart attack or stroke.

The study is published in the journal PNAS.

Reprogramming a Heartbeat

Image: Flickr - Shannon Yeh
Once damaged, heart muscle has very limited capacity for regeneration but scientists at the Gladstone Institute of Cardiovascular Diseases have now discovered how to reprogram structural fibroblasts into functioning cardiac muscle cells (cardiomyocytes). The team explored the effects of transcription factors known to be important for development of the heart and found that a combination of just three (Gata4, Mef2c and Tbx5) was sufficient to rapidly and efficiently convert cardiac or dermal fibroblasts into contractile cardiomyocyte-like cells.

Gladstone scientists have previously converted mouse mesoderm – germ tissue from very early embryos – into cardiomyocytes and have reprogrammed adult cells into induced pluripotent cells which can then be converted into other cell types but, in the present study, adult cells have been directly reprogrammed into a different type of cell without involvement of a progenitor cell state. The team hope that going directly from one adult cell type to another might eliminate some of the perceived risks associated with the use of stem cells and that it will be possible to identify small molecules that are able to trigger the conversion. Although the technique has yet to be tested in human cells, and further refinement and characterisation of the reprogramming process will be needed, the heart has a large pool of fibroblasts which provide a potential source for regenerative treatments if they could be directly reprogrammed to beating cardiomyocytes.

The study is published in the journal Cell.

Oestrogen – Better Out than In for Cardiovascular Health

No Entry
Image: Flickr - GanMed64
Although oestrogen replacement lowers cardiovascular risk in post-menopausal women, treatment is associated with an increased risk of uterine and breast cancer.

The increased cancer risk is linked to oestrogen’s action at nuclear receptors but researchers at UT Southwestern Medical Center have now found that a subpopulation of oestrogen receptors outside the cell nucleus mediate the beneficial cardiovascular effects. The extra-nuclear receptors in endothelial cells are important for blood vessel maintenance and repair and also regulate production of nitric oxide which has a number of beneficial cardiovascular effects. The team have found that an oestrogen-macromolecule complex which is excluded from the nucleus is highly effective in stimulating the extra-nuclear receptors. Similar dendrimer conjugates have been successfully used as drug delivery device in animal models and the oestrogen complex was shown to provide cardiovascular protection in high cholesterol ovariectomized female mice without stimulating growth of breast or uterine cancer. The team believe that such oestrogen-macromolecule complexes could provide cardiovascular protection for both men and women and are creating molecules that may be suitable for use in humans.

The study is published in the Journal of Clinical Investigation.

Regulating Cholesterol Levels

Image: Flickr – David Masters
Cholesterol is an essential component of all cellular membranes and is also required for synthesis of vitamin D and steroid hormones. Since it is poorly soluble in water, it is mainly transported through the bloodstream within lipoproteins – complex spherical particles composed of amphiphilic proteins and lipids whose outward-facing surfaces are water-soluble and inward-facing surfaces are lipid-soluble. Triglycerides and cholesterol esters are carried internally whilst phospholipids and cholesterol are transported in the surface monolayer of the lipoprotein particle. Several types of lipoproteins are found in blood, comprised of different apolipoproteins (which target specific tissues via receptor recognition) and with different capacities for cholesterol. These are usually referred to by their densities – the higher the ratio of cholesterol to lipoprotein, the lower the density. This gives rise to the so-called “bad cholesterol” (low density lipoprotein, LDL-cholesterol) and “good cholesterol” (HDL-cholesterol).

cholesterol structure
Problems arise when levels of the various lipoproteins are out of balance. Increased circulating levels of LDL-cholesterol are associated with the formation of foam cells, which can become trapped in the walls of blood vessels and contribute to artherosclerotic plaque formation leading to heart attacks and strokes. Conversely, HDL transports lipids to the liver for disposal and removes cholesterol from peripheral tissues, including the foam cells that form atherosclerotic plaques.

A new study by researchers at Massachusetts General Hospital (MGH) has now identified micro RNAs (miRNAs) that appear to play an important role in regulation of cholesterol/lipid levels. The team found that two members of the miR-33 family (miR-33a and miR-33b) target the ATP-binding cassette transporter A1 (ABCA1), an important regulator of HDL synthesis and reverse cholesterol transport, for posttranscriptional repression. Using antisense inhibition of miR-33 in mouse and human cell lines the researchers demonstrated up-regulation of ABCA1 expression and increased cholesterol efflux. Further, treatment of mice on a western-type diet with the antisense inhibitor resulted in elevated plasma HDL without affecting levels of LDL. The findings suggest that miR-33 may represent a therapeutic target for cardiovascular diseases.

The study is published in Science.

Chymase Inhibitors Could Improve Outcomes for Heart Failure Patients

Frozen Heart
Image: Flickr - laradanielle
Angiotensin converting enzyme (ACE) inhibitors are widely used for the treatment of hypertension and heart failure. The beneficial effects of ACE inhibitors in heart failure are believed to be linked to reduced local production of the potent vasopressor, angiotensin II (AII), but the realisation that another enzyme, chymase, can also produce AII suggests that chronic ACE inhibitor treatment may not completely suppress production of AII.

Chymase is a serine protease found mainly in mast cells but also in the cardiac interstitial space and in some cardiac endothelial cells. Scientists at Emory University, the University of Alabama, Birmingham, and Fukuoka University have now shown that adding a chymase inhibitor to ACE inhibitor treatment can significantly improve recovery of heart function in animals following a heart attack.

The team showed that chronic ACE inhibition produced a bradykinin-dependent release of a chymase (mouse mast cell protease-4, mMCP4) from mast cells in conscious mice, which maintains levels of AII in the interstitial fluid. ACE inhibition also decreases bradykinin degradation and the release of chymase into the left ventricular interstitial fluid may be the result of activation of bradykinin B2 receptors on mast cells. In hamsters, chronic ACE inhibition was found to improve left ventricular function and reduce the amount of dead tissue and scarring after myocardial infarction (MI) and the beneficial effects of chymase inhibition were greater when combined with ACE inhibition. Chymase inhibition may also affect maturation of proteases involved in tissue remodelling, independently of increases in AII levels.

Although recognising that clinical validation is still needed – and that there are no chymase inhibitors available for use in the clinic – the authors propose that addition of a chymase inhibitor to ACE inhibitor therapy may improve outcomes for patients with cardiac diseases including hypertensive heart disease, viral myocarditis, and atherosclerotic coronary artery disease, the prelude to MI.

Controlling Fat Distribution

Image: Patrick Hoesly
Extreme accumulation of fat in muscle tissue is associated with cardiovascular disease and is a contributory factor in insulin resistance and type II diabetes. It is therefore important to understand the mechanisms by which fat is taken up from the bloodstream and metabolised by tissues. Surprisingly, the role of blood vessels themselves in the transport of lipids has not been clearly established. Researchers at the Karolinska Institutet have now identified a role for vascular endothelial growth factor-B (VEGF-B) in endothelial targeting of lipids to peripheral tissues.

The VEGFs and their receptors are major regulators of angiogenesis and pharmacological intervention, for example with bevacizumab (a monoclonal antibody specific for VEGF-A), has been successfully exploited in oncology. This latest study has shown that VEGF-B, in mice, controls endothelial uptake of fatty acids via transcriptional regulation of vascular fatty acid transport proteins. Mice that were deficient in VEGF-B (Vegfb-/-) showed reduced uptake and accumulation of lipids in muscle, heart and brown adipose tissue. Instead, the Vegfb-/- mice preferentially transported lipids to white adipose tissue, resulting in a small weight increase. This regulation was mediated by VEGF receptor 1 and neuropilin 1 expressed by the endothelium.

The authors of the study, published in Nature, propose that this new role for VEGF-B could potentially lead to novel strategies to modulate pathological lipid accumulation in diabetes, obesity and cardiovascular diseases.

Role for ROCK in Cavernous Malformation

Image: Flickr – Eschipul
Cerebral cavernous malformations (CCM) are irregular clusters of dilated, leaky capillaries found in the central nervous system in around 0.5% of the general population. Although many of those with the condition will never be aware of the fact, for others the symptoms can be severe. Depending on the specific location of the CCM in the brain or spinal cord, patients may experience seizures, headaches, paralysis, hearing or vision changes, and cerebral haemorrhage. Current treatment options rely on management of the symptoms (e.g. control of seizures with anti-epileptic drugs) or surgical resection.

Researchers at University of North Carolina School of Medicine, Chapel Hill have now identified a potential target for therapeutic intervention in CCM. The disease is associated with mutations in any of three genes, ccm1, ccm2 or ccm3, which encode the corresponding CCM-1, -2 and -3 proteins. These proteins form a common complex and act co-ordinately in regulation of the cytoskeleton. It had previously been shown that loss of CCM-2 resulted in overexpression of the GTPase, RhoA, but this latest study demonstrates that CCM-1 and CCM-3 are also required for regulation of RhoA.

Y-27632 structure
The team were able to restore normal function to endothelial cells lacking CCM-1, -2 or -3 by inhibition of the RhoA-activated Rho Kinase (ROCK), either using an inhibitor, Y-27632, or shRNA knockdown of ROCK2. The results suggest that inhibition of ROCK may represent a target for pharmacological intervention in this disease.

The study is published in the Journal of Biological Chemistry.

Battle of the Bulge

Bulging Tree
Image: Flickr – zappowbang
An aortic aneurysm is a bulge in the aorta, the largest blood vessel in the body, which results from weakening of the artery wall. The majority of these occur in the portion of the aorta that passes through the abdomen and are referred to as abdominal aortic aneurysms (AAA). AAA is something of a stealth disease, since it is generally asymptomatic and may only be diagnosed at a routine physical examination or following X-ray. Over time the aneurysm may expand, with an increased risk of rupturing. Unfortunately, the rapid blood loss following aneurysm rupture is frequently fatal and accounts for at least 15,000 deaths in the US annually.

The only treatment for AAA currently available is surgical intervention. Early diagnosis is followed by monitoring the size of the aneurysm until the risk of rupture exceeds the risk of surgery. However, scientists at Providence Heart + Lung Institute at St. Paul’s Hospital and the University of British Columbia (UBC) have now raised the possibility of pharmacological intervention. Using experimental models of AAA, the team have found a role for the protein-degrading enzyme Granzyme B (GMZB).

GMZB is a serine protease expressed by a variety of immune cells and is responsible for destroying unwanted tissue, such as virally-infected cells. This role is supported by the pore-forming protein, perforin, which delivers GMZB to the intracellular compartment. The UBC research has shown that GMZB, which is abundantly expressed in aneurysms from human and animal model AAA, also plays a role in the pathogenesis of AAA. Further, the experimental data suggest that this is a perforin-independent mechanism involving extracellular matrix degradation and subsequent loss of vessel wall integrity. The results suggest that an inhibitor of GMZB may provide a therapeutic option in the treatment of AAA.

The study is published in the American Journal of Pathology.

Toll-Like Receptor 2 Linked to Atherosclerotic Damage

Image: Flickr - dawnzy
Image: Flickr - dawnzy
Atherosclerosis is caused by a build up of lipids, cholesterol, calcium, and cellular debris within the artery, resulting in plaque formation. This restricts the flow of blood and decreases oxygen supply to target organs, increasing the risk of cardiovascular diseases including heart attacks and stroke. The true incidence of atherosclerosis is difficult, if not impossible, to determine since it is predominantly asymptomatic, but the ensuing cardiovascular diseases are the leading cause of death in many Western societies. Risk factors for atherosclerosis include high blood pressure and a high fat diet and current non-surgical treatments, such as antihypertensive medicines and statins, focus on reducing these risks.

The precise mechanisms underlying atherogenesis are unclear but scientists at Imperial College London have now identified a pathway that plays a key role in the inflammation and matrix degradation characteristic of human atherosclerosis.

The researchers studied sections of carotid artery taken from 58 stroke patients and found that toll-like receptor 2 (TLR-2) was unusually active in the plaques. TLRs are expressed on immune cells and play a fundamental role in pathogen recognition and innate immunity, mediating release of cytokines and other inflammatory mediators. One arm of the TLR-induced inflammatory response is dependent on a signalling pathway that is mediated by the adaptor molecule, myeloid differentiation primary response gene 88 (MyD88), and the study showed that a dominant-negative form of MyD88 decreased the production of MCP-1, IL-8, IL-6, MMP-1 and MMP-3 as well as NF-κB activation in cell cultures prepared from the carotid arteries. TLR-2 neutralizing antibodies were also shown to inhibit NF-κB activation and significantly reduce MCP-1, IL-8, IL-6, MMP-1, MMP-2, MMP-3, and MMP-9 production. In contrast, an IL-1R antagonist, TLR-4 blocking antibodies, or overexpression of a dominant-negative form of the TLR-4 signalling adaptor, TRIF-related adaptor molecule, reduced NF-κB activity but did not have a broad impact on the production of the inflammatory mediators studied.

The authors hope that TLR-2 blockers might be developed to prevent or treat atherosclerosis and the resulting cardiovascular disease without compromising the ability to fight infection.

The study is published in the journal Circulation.

New Drug Target for Pulmonary Arterial Hypertension

Image: Flickr - Jemsweb
Image: Flickr - Jemsweb
Pulmonary arterial hypertension (PAH) is a progressive, debilitating disease characterised by increased resistance in pulmonary arteries, placing additional workload on the right ventricle of the heart. Untreated, the disease frequently results in right ventricular failure and death. Until the 1990s the only effective treatment was heart-lung transplantation. Subsequently, drug treatments that have been used include anticoagulants, calcium channel blockers, prostacylin and endothelin receptor antagonists. Whilst these drugs have demonstrated efficacy in PAH patients, delaying the need for lung transplantation, long term survival rates have not been significantly impacted.

The annual incidence of PAH is around 1-2 per million individuals, with a further 8 per million contributed by PAH associated with scleroderma. Despite the low incidence, there are approximately 100,000 PAH patients in Europe and the US.

Scientists at University of California–San Diego (UCSD) have now established that human pulmonary hypertension is characterised by overexpression of Notch3 in small pulmonary artery smooth muscle cells and that the severity of disease in humans and rodents correlates with the amount of Notch3 protein in the lung. In the study, published online in Nature Medicine on 25th October, the team showed that mice with homozygous deletion of Notch3 do not develop pulmonary hypertension in response to hypoxic stimulation. Additionally, mice with pulmonary hypertension were successfully treated with a DAPT, a γ-secretase inhibitor that blocks activation of Notch3.

Notch receptor signalling is implicated in control of smooth muscle cell proliferation and maintaining smooth muscle cells in an undifferentiated state. The discovery that the Notch3 signalling pathway is crucial for the development of PAH provides a novel target for therapeutic intervention.