A-NO-ther Incarnation for COX Inhibitors?

Image: Rishart3
Selective cyclooxygenase-2 (COX-2) inhibitors were developed to reduce the risk of gastrointestinal side effects associated with the older non-steroidal anti-inflammatory drugs (NSAIDs) which inhibit both COX-1 and COX-2. The withdrawal of rofecoxib (Vioxx™) in 2004 and valdecoxib (Bextra™) in 2005 because of a slightly increased risk of thrombotic events such as heart attacks and strokes after long-term treatment came as a blow to the pharmaceutical industry, doctors and patients. Since the withdrawal of rofecoxib and valdecoxib, no new anti-inflammatory drugs have been submitted to the FDA for the treatment of osteoarthritis.

Last month, however, French pharmaceutical company, NicOX, submitted an NDA for its first-in-class cyclooxygenase-inhibiting nitric oxide donator (CINOD), naproxcinod, for the treatment of osteoarthritis.

Naproxcinod is a nitroxybutyl ester of the well established and well tolerated NSAID, naproxen, and is intended to overcome another drawback of NSAIDs and COX-2 inhibitors – increased blood pressure. Cleavage of naproxcinod by esterases gives naproxen, together with a nitric oxide donating moiety. Nitric oxide relaxes vascular smooth muscle cells, causing dilation of the arteries and reducing blood pressure, and has also been reported to reduce formation of thrombi. In clinical trials in patients with osteoarthritis of the knee and hip, naproxcinod met efficacy endpoints and did not cause an increase in blood pressure. Naproxcinod also showed an advantage compared with naproxen in terms of gastrointestinal side effects.

PDE1a Linked to Heart Failure

Sildenafil also significantly prolongs the time before cut flowers wilt
Sildenafil also significantly prolongs the time before cut flowers wilt.

Photo: Flickr: AbiznessDigital

Although sildenafil (Viagra®) is best known for the treatment of erectile dysfunction, it also reduces pulmonary hypertension and its use in the treatment of heart failure and cardiac hypertrophy is being investigated.

Sildenafil is a selective inhibitor of phosphodiesterase type 5 (PDE5) which enhances nitric oxide-mediated vasodilation by preventing breakdown of the intracellular second messenger, cyclic guanosine monophosphate (cGMP). Phosphodiesterases regulate cGMP-dependent signalling pathways including control of Ca2+ concentrations which, as well as modulating the strength of contractions, are believed to regulate myocyte growth and hypertrophy. At least five PDE families (PDE1-5) are expressed in the human heart and a study led by researchers at the University of Rochester Medical Center has now shown that the Ca2+/calmodulin-activated phosphodiesterase type 1 (PDE1) enzymes play a major role in cardiac disease.

Either down-regulation of PDE1 using siRNA or treatment with the selective inhibitor, IC86340, prevented phenylephrine-induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal rat ventricular myocytes and adult rat ventricular myocytes. Studies in mice showed that IC86340 also reduced the cardiac hypertrophy caused by chronic infusion of isoproterenol. Although both PDE1a and PDE1c mRNA and protein were detected in human hearts, only PDE1a was found in rodent hearts. PDE1a is also up-regulated in heart tissue from various animals that model pathological hypertrophy, suggesting that PDE1a plays a key role in mediating pathological cardiomyocyte hypertrophy. Studies in isolated myocytes showed that a combination of sildenafil and IC86340 reduced hypertrophy more than either compound alone and the team now plan to explore the effects of combination treatment in animal studies.

The study is published in the journal Circulation Research.

MMP8 is Major Player in Atherosclerosis

Coronary artery with complex atherosclerosis and luminal narrowing.  Image: Nephron
Coronary artery with complex atherosclerosis and luminal narrowing.

Image: Nephron

Both high blood pressure and atherosclerosis, a condition in which the arteries become narrowed by atheroma, are major risk factors for cardiovascular diseases, including heart attack and stroke. The atheromatous plaques are made up of cells (mostly macrophages) or cell debris containing oxidised lipids, calcium and connective tissue.

A team led by scientists in the UK has now shown that matrix metalloproteinase-8 (MMP8) plays a crucial role both in raising blood pressure, and in causing the abnormal build-up of cells and fatty deposits in the arteries. MMPs are best known for their ability to degrade extracellular matrix proteins such as collagen, but have also been shown to cleave a variety of other proteins. As well as degrading collagen type 1, MMP8 has been shown to cleave angiotensin I to produce the vasopressor, angiotensin II. This conversion is also carried out by angiotensin converting enzyme (ACE) and ACE inhibitors are commonly prescribed to treat patients with high blood pressure and congestive heart failure as well as those who have suffered a heart attack. In the present study, the team found that inactivating MMP8 in apolipoprotein E-deficient mice – which are prone to atherosclerosis – resulted in a substantial reduction in atherosclerotic lesions and that the lesions had fewer macrophages but increased collagen content. The knockout mice were also found to have reduced levels of angiotensin II and lower blood pressure, as well as reduced expression of vascular cell adhesion molecule (VCAM)-1 in atherosclerotic lesions.

The team also evaluated data from a group of 2000 patients with coronary artery disease who were undergoing a coronary angiogram procedure and found an association between an MMP8 gene variant and degree of atherosclerosis. The researchers hope that their findings will lead to new drugs to treat hypertension and prevent heart disease, particularly for patients who fail to respond well to ACE inhibitors.

The study is published in the journal Circulation Research.

The Renin-Angiotensin System and MS

The renin-angiotensin system (RAS) was first studied for its role in regulation of the cardiovascular system and drugs that modulate the RAS are now widely used to treat high blood pressure, myocardial infarction and stroke. More recently, it has become apparent that components of the RAS also mediate inflammatory processes and two recently published studies have now expanded on the link between the RAS and multiple sclerosis (MS). A team led by researchers at Stanford University School of Medicine found that multiple sclerosis lesions from brains of MS patients had elevated levels of both the angiotensin I receptor (AT1R) and angiotensin converting enzyme (ACE). The team then showed that treatment with the ACE inhibitor, lisinopril, or the AT1R antagonist, candesartan, could prevent the development of experimental autoimmune encephalomyelitis (EAE) in mice and, perhaps more importantly, reverse the symptoms of established disease. Reduced activation of AT1R was shown to increase the number of Treg cells in the CNS and suppress TH1/TH17-mediated immune responses to autoantigens.
renin angiotensin system scheme
The study is published in the online early edition of PNAS.

The second study, by researchers in Germany and also published in the online early edition of PNAS, showed that renin, ACE and AT1R were all up-regulated in the inflamed spinal cord and immune system, including antigen presenting cells (APC), of mice with EAE. Pretreatment with the renin inhibitor, aliskiren; the ACE inhibitor, enalapril; or the AT1R antagonist, losartan, reduced the severity of EAE symptoms and losartan was also found to ameliorate the course of established disease. Blockade of AT1R was found not to have a direct effect on T-cell responses but to significantly reduce APC in the spinal cord and immune organs, and to reduce cytokine-induced APC migration.

Since drugs that modulate the RAS have been used in millions of people around the world and have few side effects, the researchers hope that clinical trials to test their effectiveness in MS patients should be straightforward to carry out.

New Hope for Damaged Hearts

Scars of HeartA number of organs, including the heart, have limited regenerative powers, but US scientists have now shown that fully differentiated cardiac muscle cells can be induced to proliferate and regenerate. Writing in the journal Cell, they show that the growth factor neuregulin 1, which plays a role in early development of the heart and nervous system, induces mononucleated, but not binucleated, cardiomyocytes to divide in vitro by acting on the receptor tyrosine kinase, ErbB4. In mice, genetic inactivation of ErbB4 was shown to reduce cardiomyocyte proliferation, whereas increasing ErbB4 expression enhanced proliferation. Following heart attack in adult mice, daily intraperitoneal injection of neuregulin 1 for 12 weeks led to regeneration of the heart muscle and improved function. Unlike the control mice, the treated animals showed reduced signs of heart failure such as left-ventricular dilation and cardiac hypertrophy. If the neuregulin/ErbB4 signalling pathway plays the same role in human heart muscle, stimulating proliferation of differentiated cardiomyocytes by activation of this pathway may provide an alternative to stem cell therapy to regenerate damaged heart muscle in patients with heart failure or children with congenital heart defects. Since ErbB receptor tyrosine kinases and neuregulins have oncogenic potential and may cause proliferation of other tissues, a full safety assessment would be needed before any clinical studies.

Neuregulin 1 has previously been associated with susceptibility to schizophrenia and has also been shown to protect neurones following stroke.

Renin Links CMV Infection to High Blood Pressure

barometerHuman cytomegalovirus (hCMV) is a widespread member of the herpes family of viruses, with well over half of the world’s adult population thought to be infected. Most individuals are infected in early childhood and remain infected with latent virus for the rest of their lives. For the majority of people, infection is asymptomatic and usually undiagnosed but immunosuppression, caused by HIV-1 infection or drug therapy after transplant surgery, can lead to reactivation of the virus and overt infection. A number of studies have linked hCMV infection to cardiovascular disease, but the mechanisms underlying the pathology are not well understood. Writing in the May 15th edition of PLoS Pathogens, a team led by researchers at Beth Israel Deaconess Medical Center have now shown that infection with murine CMV (mCMV) leads to a significant increase in arterial pressure in mice. mCMV infection alone was sufficient to cause the observed increase in blood pressure. mCMV infection alone did not cause atherosclerosis in the aorta but, when combined with a high cholesterol diet, did cause classic plaque formation. The team went on to show that mCMV stimulated production of pro-inflammatory cytokines, IL6, TNF-α and MCP-1 which have previously been linked to high blood pressure. They also showed that mCMV infection induced renin expression in an infection dose-related manner in mouse renal cells and that hCMV induced a similar increase in human vascular endothelial cells. In mice, mCMV infection was additionally shown to lead to an increase in angiotensin II levels in serum and in tissue from the aorta.

Since the roles of renin and angiotensin II in hypertension are well established, the study provides a mechanism by which persistent CMV infection might increase blood pressure and also suggests that vaccination or antiviral therapy could have the potential to provide new treatments for cardiovascular disease.

New Target for Treating Cardiovascular Disease

Researchers from University of Rochester Medical Center have shown that eliminating the gene for cyclophilin A completely protects mice from developing abdominal aortic aneurysms, a late stage complication of atherosclerosis. An aortic aneurysm is a thin, weakened section of the aorta which can rupture, leading to massive internal blood loss and death. Aneurysms occur most frequently in the abdominal section of the aorta and cause around 15,000 deaths a year, most in older men. In abdominal aortic aneurysm, angiotensin II is known to stimulate oxidative stress in blood vessels leading to increased activity of matrix metalloproteinases which, in turn, degrade the matrix structure of the vessel wall. Increased activity of matrix metalloproteinases also plays a role in atherosclerosis, allowing smooth muscle cells from the blood vessel walls to contribute to the development of plaques. In both abdominal aortic aneurysm and atherosclerosis, angiotensin II also contributes to local inflammation by recruiting immune cells to the blood vessel wall.

aortic aneurysmUsing genetically modified mice, cyclophilin A was found to promote all three events involved in angiotensin II mediated damage to blood vessels – oxidative stress, matrix degradation and inflammation. Cyclophilin A is highly expressed in vascular smooth muscle cells and studies showed that both intracellular and extracellular cyclophilin A are required for generation of reactive oxygen species and activation of matrix metalloproteinases. The team is hoping to develop anti-cyclophilin A drugs that will reduce the processes underlying cardiovascular diseases such as abdominal aortic aneurysm, atherosclerosis and hypertension. The study is published in full in the journal Nature Medicine.

New Blood Pressure Target

heartAlthough normally clinically silent, persistent hypertension and atherosclerosis are leading risk factors for strokes, heart attacks, heart failure, aneurysms and chronic renal failure. How blood pressure is regulated is still not fully understood, but a team lead by scientists at the University of Pennsylvania School of Medicine has now suggested a role for prostaglandin F2α (PGF2α) in increasing blood pressure and accelerating atherosclerosis. Prostaglandins are a group of hormone-like substances that mediate many physiological and pathophysiological processes, and the team found that mice lacking the receptor for PGF2α had lower blood pressure and less atherosclerosis than wild-type mice. Knocking out the PGF2α receptor was found to suppress activity of the renin-angiotensin system which plays a key role in regulating blood pressure. When blood pressure is low, the liver secretes a protein, angiotensinogen, which is cleaved by renin to give angiotensin I. Further cleavage by angiotensin converting enzyme (ACE) produces angiotensin II which increases blood pressure by narrowing blood vessels and by stimulating release of aldosterone which leads in turn to retention of sodium and water.

If the results seen in mice translate to humans, blocking the PGF2α receptor may provide a novel strategy for controlling blood pressure and reducing atherosclerosis. The study is published in full in the Proceedings of the National Academy of Sciences.

Mending a Broken Heart

mending a broken heartHeart disease is a leading cause of death and illness in the developed world and, once damaged, the heart has very limited capacity for regeneration. Following a heart attack, if blood flow is not restored to the heart muscle within 20-40 minutes, the muscle cells (cardiomyocytes) will die. The dead cells are replaced by scar tissue which does not contract or pump as well as healthy heart tissue.

Writing in the journal Nature, Jun Takeuchi and Benoit Bruneauat at the Gladstone Institute of Cardiovascular Disease have now identified a cocktail of three proteins that can turn mouse mesoderm into cardiac muscle cells (cardiomyocytes). Mesoderm is one of the three primary germ cell layers in the very early embryo – the others are the ectoderm and the endoderm – that can differentiate to give a number of tissues such as bone, blood, and muscle, including heart muscle. The three key proteins are the cardiac transcription factors, GATA4 and TBX5, which are believed to be involved in heart development and function, and a cardiac-specific subunit of BAF chromatin-remodelling complexes, Baf60c. Defects in the genes for these proteins have been linked to abnormal development and defects in the heart.

A combination of all three proteins was shown to direct differentiation of mouse mesoderm specifically into cardiac muscle cardiomyocytes that beat rhythmically, just like normal heart cells. Although, so far, only cells from very early mouse embryos have been turned into cardiomyocytes, Takeuchi and Bruneauat hope that their work will help to understand how new cardiomyocytes can be produced for use in regenerative medicine to treat heart disease.

Making Light Work of Blood Clotting

traffic lightThe ability to selectively switch enzymes on and off in a particular tissue could offer many improved treatment options, and scientists at the University of Florida have now devised a way of achieving this goal. Writing in the journal PNAS, they describe a molecule consisting of a DNA aptamer attached by a polyethylene glycol linker to a complementary strand of DNA with azobenzene molecules attached. The aptamer was chosen to bind selectively to the enzyme thrombin and block its role in blood coagulation. In visible light, the azobenzene double bonds are in the trans-conformation and the aptamer is prevented from binding to thrombin by hybridisation with the complementary strand of DNA. When irradiated with ultraviolet light, however, the azobenzene molecules flip into the cis-conformation causing dissociation of the duplex and unveiling the aptamer which can then bind to thrombin and inhibit blood clotting.

The team hope that the technique could one day be used to cut off the blood supply to solid tumours or to produce controlled release versions of drugs which would be activated only in the target tissue, thus reducing the risk of side effects. Endoscopic lights could be used to activate the drugs or, in some cases, it may be sufficient to irradiate skin close to the target site with near-infrared light which penetrates the skin.