Targeted delivery of therapeutics to bone and connective tissues: current status and challenges- Part II

 

Curator/Reporter: Aviral Vatsa PhD MBBS

This post is in the second part of the reviews that focuses on the current status of drug delivery to bone and the issues facing this field. The first part can be accessed here

Annual treatment costs for musculoskeletal diseases in the US are roughly 7.7% (~ $849 billion) of total gross domestic product. Such disorders are the main cause of physical disability in US. Almost half of all chronic conditions in people can be attributed to bone and joint disorders. In addition there is increasing ageing population and associated increases in osteoporosis and other diseases, rising incidences of degenerative intervertebral disk diseases and numbers of revision orthopedic arthroplasty surgeries, and increases in spinal fusions. All these factors contribute towards the increasing requirement of bone regeneration and reconstruction methods and products. Delivery of therapeutic grade products to bone has various challenges. Parenteral administration limits the efficient delivery of drugs to the required site of injury and local delivery methods are often expensive and invasive. The theme issue of Advance Drug Delivery reviews focuses on the current status of drug delivery to bone and the issues facing this field. Here is the second part of these reviews and research articles.

1. Targeting polymer therapeutics to bone [1]

Abstract

An aging population in the developing world has led to an increase in musculoskeletal diseases such as osteoporosis and bone metastases. Left untreated many bone diseases cause debilitating pain and in the case of cancer, death. Many potential drugs are effective in treating diseases but result in side effects preventing their efficacy in the clinic. Bone, however, provides a unique environment of inorganic solids, which can be exploited in order to effectively target drugs to diseased tissue. By integration of bone targeting moieties to drug-carrying water-soluble polymers, the payload to diseased area can be increased while side effects decreased. The realization of clinically relevant bone targeted polymer therapeutics depends on (1) understanding bone targeting moiety interactions, (2) development of controlled drug delivery systems, as well as (3) understanding drug interactions. The latter makes it possible to develop bone targeted synergistic drug delivery systems.

2. Development of macromolecular prodrug for rheumatoid arthritis [2]

Abstract

Rheumatoid arthritis (RA) is a chronic autoimmune disease that is considered to be one of the major public health problems worldwide. The development of therapies that target tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and co-stimulatory pathways that regulate the immune system have revolutionized the care of patients with RA. Despite these advances, many patients continue to experience symptomatic and functional impairment. To address this issue, more recent therapies that have been developed are designed to target intracellular signaling pathways involved in immunoregulation. Though this approach has been encouraging, there have been major challenges with respect to off-target organ side effects and systemic toxicities related to the widespread distribution of these signaling pathways in multiple cell types and tissues. These limitations have led to an increasing interest in the development of strategies for the macromolecularization of anti-rheumatic drugs, which could target them to the inflamed joints. This approach enhances the efficacy of the therapeutic agent with respect to synovial inflammation, while markedly reducing non-target organ adverse side effects. In this manuscript, we provide a comprehensive overview of the rational design and optimization of macromolecular prodrugs for treatment of RA. The superior and the sustained efficacy of the prodrug may be partially attributed to their Extravasation through Leaky Vasculature and subsequent Inflammatory cell-mediated Sequestration (ELVIS) in the arthritic joints. This biologic process provides a plausible mechanism, by which macromolecular prodrugs preferentially target arthritic joints and illustrates the potential benefits of applying this therapeutic strategy to the treatment of other inflammatory diseases.

3. Peptide-based delivery to bone [3]

Abstract

Peptides are attractive as novel therapeutic reagents, since they are flexible in adopting and mimicking the local structural features of proteins. Versatile capabilities to perform organic synthetic manipulations are another unique feature of peptides compared to protein-based medicines, such as antibodies. On the other hand, a disadvantage of using a peptide for a therapeutic purpose is its low stability and/or high level of aggregation. During the past two decades, numerous peptides were developed for the treatment of bone diseases, and some peptides have already been used for local applications to repair bone defects in the clinic. However, very few peptides have the ability to form bone themselves. We herein summarize the effects of the therapeutic peptides on bone loss and/or local bone defects, including the results from basic studies. We also herein describe some possible methods for overcoming the obstacles associated with using therapeutic peptide candidates.

4. Growth factor delivery: How surface interactions modulate release in vitro and in vivo [4]

Abstract

Biomaterial scaffolds have been extensively used to deliver growth factors to induce new bone formation. The pharmacokinetics of growth factor delivery has been a critical regulator of their clinical success. This review will focus on the surface interactions that control the non-covalent incorporation of growth factors into scaffolds and the mechanisms that control growth factor release from clinically relevant biomaterials. We will focus on the delivery of recombinant human bone morphogenetic protein-2 from materials currently used in the clinical practice, but also suggest how general mechanisms that control growth factor incorporation and release delineated with this growth factor could extend to other systems. A better understanding of the changing mechanisms that control growth factor release during the different stages of preclinical development could instruct the development of future scaffolds for currently untreatable injuries and diseases.

5. Biomaterial delivery of morphogens to mimic the natural healing cascade in bone[5]

Abstract

Complications in treatment of large bone defects using bone grafting still remain. Our understanding of the endogenous bone regeneration cascade has inspired the exploration of a wide variety of growth factors (GFs) in an effort to mimic the natural signaling that controls bone healing. Biomaterial-based delivery of single exogenous GFs has shown therapeutic efficacy, and this likely relates to its ability to recruit and promote replication of cells involved in tissue development and the healing process. However, as the natural bone healing cascade involves the action of multiple factors, each acting in a specific spatiotemporal pattern, strategies aiming to mimic the critical aspects of this process will likely benefit from the usage of multiple therapeutic agents. This article reviews the current status of approaches to deliver single GFs, as well as ongoing efforts to develop sophisticated delivery platforms to deliver multiple lineage-directing morphogens (multiple GFs) during bone healing.

6. Studies of bone morphogenetic protein-based surgical repair[6]

Abstract

Over the past several decades, recombinant human bone morphogenetic proteins (rhBMPs) have been the most extensively studied and widely used osteoinductive agents for clinical bone repair. Since rhBMP-2 and rhBMP-7 were cleared by the U.S. Food and Drug Administration for certain clinical uses, millions of patients worldwide have been treated with rhBMPs for various musculoskeletal disorders. Current clinical applications include treatment of long bone fracture non-unions, spinal surgeries, and oral maxillofacial surgeries. Considering the growing number of recent publications related to clincal research of rhBMPs, there exists enormous promise for these proteins to be used in bone regenerative medicine. The authors take this opportunity to review the rhBMP literature paying specific attention to the current applications of rhBMPs in bone repair and spine surgery. The prospective future of rhBMPs delivered in combination with tissue engineered scaffolds is also reviewed.

7. Strategies for controlled delivery of growth factors and cells for bone regeneration[7]

Abstract

The controlled delivery of growth factors and cells within biomaterial carriers can enhance and accelerate functional bone formation. The carrier system can be designed with pre-programmed release kinetics to deliver bioactive molecules in a localized, spatiotemporal manner most similar to the natural wound healing process. The carrier can also act as an extracellular matrix-mimicking substrate for promoting osteoprogenitor cellular infiltration and proliferation for integrative tissue repair. This review discusses the role of various regenerative factors involved in bone healing and their appropriate combinations with different delivery systems for augmenting bone regeneration. The general requirements of protein, cell and gene therapy are described, with elaboration on how the selection of materials, configurations and processing affects growth factor and cell delivery and regenerative efficacy in both in vitro and in vivo applications for bone tissue engineering.

8. Bone repair cells for craniofacial regeneration[8]

Abstract

Reconstruction of complex craniofacial deformities is a clinical challenge in situations of injury, congenital defects or disease. The use of cell-based therapies represents one of the most advanced methods for enhancing the regenerative response for craniofacial wound healing. Both somatic and stem cells have been adopted in the treatment of complex osseous defects and advances have been made in finding the most adequate scaffold for the delivery of cell therapies in human regenerative medicine. As an example of such approaches for clinical application for craniofacial regeneration, Ixmyelocel-T or bone repair cells are a source of bone marrow derived stem and progenitor cells. They are produced through the use of single pass perfusion bioreactors for CD90+ mesenchymal stem cells and CD14+ monocyte/macrophage progenitor cells. The application of ixmyelocel-T has shown potential in the regeneration of muscular, vascular, nervous and osseous tissue. The purpose of this manuscript is to highlight cell therapies used to repair bony and soft tissue defects in the oral and craniofacial complex. The field at this point remains at an early stage, however this review will provide insights into the progress being made using cell therapies for eventual development into clinical practice.

9. Gene therapy approaches to regenerating bone[9]

Abstract

Bone formation and regeneration therapies continue to require optimization and improvement because many skeletal disorders remain undertreated. Clinical solutions to nonunion fractures and osteoporotic vertebral compression fractures, for example, remain suboptimal and better therapeutic approaches must be created. The widespread use of recombinant human bone morphogenetic proteins (rhBMPs) for spine fusion was recently questioned by a series of reports in a special issue of The Spine Journal, which elucidated the side effects and complications of direct rhBMP treatments. Gene therapy – both direct (in vivo) and cell-mediated (ex vivo) – has long been studied extensively to provide much needed improvements in bone regeneration. In this article, we review recent advances in gene therapy research whose aims are in vivo or ex vivo bone regeneration or formation. We examine appropriate vectors, safety issues, and rates of bone formation. The use of animal models and their relevance for translation of research results to the clinical setting are also discussed in order to provide the reader with a critical view. Finally, we elucidate the main challenges and hurdles faced by gene therapy aimed at bone regeneration as well as expected future trends in this field.

10. Gene delivery to bone[10]

Abstract

Gene delivery to bone is useful both as an experimental tool and as a potential therapeutic strategy. Among its advantages over protein delivery are the potential for directed, sustained and regulated expression of authentically processed, nascent proteins. Although no clinical trials have been initiated, there is a substantial pre-clinical literature documenting the successful transfer of genes to bone, and their intraosseous expression. Recombinant vectors derived from adenovirus, retrovirus and lentivirus, as well as non-viral vectors, have been used for this purpose. Both ex vivo and in vivo strategies, including gene-activated matrices, have been explored. Ex vivo delivery has often employed mesenchymal stem cells (MSCs), partly because of their ability to differentiate into osteoblasts. MSCs also have the potential to home to bone after systemic administration, which could serve as a useful way to deliver transgenes in a disseminated fashion for the treatment of diseases affecting the whole skeleton, such as osteoporosis orosteogenesis imperfecta. Local delivery of osteogenic transgenes, particularly those encoding bone morphogenetic proteins, has shown great promise in a number of applications where it is necessary to regenerate bone. These include healing large segmental defects in long bones and the cranium, as well as spinal fusion and treating avascular necrosis.

11. RNA therapeutics targeting osteoclast-mediated excessive bone resorption[11]

Abstract

RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing technique developed with dramatically increasing utility for both scientific and therapeutic purposes. Short interfering RNA (siRNA) is currently exploited to regulate protein expression relevant to many therapeutic applications, and commonly used as a tool for elucidating disease-associated genes. Osteoporosis and their associated osteoporotic fragility fractures in both men and women are rapidly becoming a global healthcare crisis as average life expectancy increases worldwide. New therapeutics are needed for this increasing patient population. This review describes the diversity of molecular targets suitable for RNAi-based gene knock down in osteoclasts to control osteoclast-mediated excessive bone resorption. We identify strategies for developing targeted siRNA delivery and efficient gene silencing, and describe opportunities and challenges of introducing siRNA as a therapeutic approach to hard and connective tissue disorders.

Bibliography

[1] S. A. Low and J. Kopeček, “Targeting polymer therapeutics to bone,” Advanced Drug Delivery Reviews, vol. 64, no. 12, pp. 1189–1204, Sep. 2012.

[2] F. Yuan, L. Quan, L. Cui, S. R. Goldring, and D. Wang, “Development of macromolecular prodrug for rheumatoid arthritis,” Advanced Drug Delivery Reviews, vol. 64, no. 12, pp. 1205–1219, Sep. 2012.

[3] K. Aoki, N. Alles, N. Soysa, and K. Ohya, “Peptide-based delivery to bone,” Advanced Drug Delivery Reviews, vol. 64, no. 12, pp. 1220–1238, Sep. 2012.

[4] W. J. King and P. H. Krebsbach, “Growth factor delivery: How surface interactions modulate release in vitro and in vivo,” Advanced Drug Delivery Reviews, vol. 64, no. 12, pp. 1239–1256, Sep. 2012.

[5] M. Mehta, K. Schmidt-Bleek, G. N. Duda, and D. J. Mooney, “Biomaterial delivery of morphogens to mimic the natural healing cascade in bone,” Advanced Drug Delivery Reviews, vol. 64, no. 12, pp. 1257–1276, Sep. 2012.

[6] K. W.-H. Lo, B. D. Ulery, K. M. Ashe, and C. T. Laurencin, “Studies of bone morphogenetic protein-based surgical repair,” Advanced Drug Delivery Reviews, vol. 64, no. 12, pp. 1277–1291, Sep. 2012.

[7] T. N. Vo, F. K. Kasper, and A. G. Mikos, “Strategies for controlled delivery of growth factors and cells for bone regeneration,” Advanced Drug Delivery Reviews, vol. 64, no. 12, pp. 1292–1309, Sep. 2012.

[8] G. Pagni, D. Kaigler, G. Rasperini, G. Avila-Ortiz, R. Bartel, and W. V. Giannobile, “Bone repair cells for craniofacial regeneration,” Advanced Drug Delivery Reviews, vol. 64, no. 12, pp. 1310–1319, Sep. 2012.

[9] N. Kimelman Bleich, I. Kallai, J. R. Lieberman, E. M. Schwarz, G. Pelled, and D. Gazit, “Gene therapy approaches to regenerating bone,” Advanced Drug Delivery Reviews, vol. 64, no. 12, pp. 1320–1330, Sep. 2012.

[10] C. H. Evans, “Gene delivery to bone,” Advanced Drug Delivery Reviews, vol. 64, no. 12, pp. 1331–1340, Sep. 2012.

[11] Y. Wang and D. W. Grainger, “RNA therapeutics targeting osteoclast-mediated excessive bone resorption,” Advanced Drug Delivery Reviews, vol. 64, no. 12, pp. 1341–1357, Sep. 2012.

 

Discovery of nitric oxide and its role in vascular biology

Curated/reported by : Aviral Vatsa PhD, MBBS

Based on : S Moncada et al

It was in 1980 that Furchgott & Zawadzki first described endothelium- dependent relaxation of the blood vessels by acetylcholine. Further studies in 1984 revealed that other factors such as bradykinin, histamine and 5-hydroxytryptamine release endothelium derived relaxing factor (EDRF), which can modulate vessel tone. EDRF was shown to stimulate soluble guanylate cyclase and was inhibited by haemoglobin. In 1986 it was demonstrated that superoxide (O₂^-) anions mediated EDRF inactivation and that the inhibitors of EDRF generated superoxide (O₂^-) anions in solution as a mean to inhibit EDRF. It was later established that all compounds that inhibit EDRF have one property in common, redox activity, which accounted for their inhibitory action on EDRF. One exception was haemoglobin, which inactivates EDRF by binding to it. In 1987 Furchgott proposed that EDRF might be nitric oxide (NO) based on a study of the transient relaxations of endothelium-denuded rings of rabbit aorta to ‘acidified’ inorganic nitrite (NO^-) solutions and the observations that superoxide dismutase (SOD, which removes O₂^-) protected EDRF. Till then NO was not known to be produced in mammalian cells. In 1988 Palmer et al could detect NO production both biologically and chemically by chemiluminescence. The following year in 1989 the enzyme responsible for NO production, NO synthase, was discovered and L-arginine:NO pathway was proposed.

Roles of L-arginine:NO pathway :

By 1987 it was proposed that NO is generated in tissues other than endothelium. Hibs et al and Marletta et al proposed that NO was generated by macrophages. Moreover release of EDRF was demonstrated in cerebellar cells following activation with N-methyl-D- aspartate (NMDA ). Both noradrenergic and cholinergic responses are ‘controlled’ by the nitrergic system so that the release of NO (e.g., during electrical field stimulation) counteracts and dominates the response to the noradrenergic or cholinergic stimulus (Cellek & Moncada, 1997). Mechanism of penile erection was unveiled by the studies on nitrergic neurotransmission that led to therapeutic intervention. Selective damage of nitrergic nerves in disease states was proposed as a potent mechanism of pathophysiology. Broadly three areas of research based on three isoforms of NOS came into being; cardiovascular, nervous and immunology. Identification of N^G-monomethyl-L-arginine (L-NMMA) as an inhibitor of the synthesis of NO lay the basis of future research into investigating the role of NO in biological systems. In 1989 it was demonstrated that intravenous infusion of L-NMMA resulted in increase in blood pressure that was reversible by infusing L-arginine. NO was thus implicated in constantly maintaining blood vessel tone. eNOS knockout studies showed a hypertensive phenotypes in the animal models and over expression of eNOS led to lowering of the blood pressure. Furthermore, eNOS activation was attributed to phosphorylation of a specific tyrosine residue in the enzyme.

NO and mitochondria: (detailed here)

NO reacts with some of the complexes of the respiratory chain, and inhibits mitochondrial respiration- this is a well accepted notion. Initially it was believed that the target for NO was soluble guanylate cyclase, which in vasculature would lead to elevation of cGMP that eventually results in NO mediated vasodilatation and platelet aggregation inhibition. In 1994, another potential target, cytochrome c oxidase, for inhibitory effects of NO was discovered. This was a reversible effect, in competition with oxygen concentrations. Increases in NO production were also shown to inhibit cellular respiration irreversibly by selectively inhibiting complex I . Hence in 2002 it was proposed that this might be a mechanism through which cell pathology was initiated in certain conditions. Furthermore, NO was proposed to be implicated in the activation of the grp78-dependent stress response , via modulating calcium -related interaction between mitochondria and endoplasmic reticulum . This host defence mechanism might also have role in vasculature. Further evidence was provided in 2003 to link the role of NO in mitochondrogenesis and thus indicating that NO might be involved in the regulation of the balance between glycolysis and oxidative phosphorylation in cells.

NO and pathophysiology :

Lack of NO: By 2000, NO was established as a haemostatic regulator in the vasculature. Its absence was implicated in pathological states such as hypertension and vasospasm. These pathophysiological states share a common beginning of endothelial dysfunction, which has low NO production as one of its characterstic features. This dysfunction has been observed prior to the appearance of cardiovascular disease in predisposed subjects with family history of essential hypertension and atherosclerosis. The most likely mechanism for endothelial dysfunction is that of a reduced bioavailability of NO . The mechanism of this aspect is discussed elsewhere on this site. Protection against reduction of NO bio-availability in the vasculature is a vital therapeutic target and is extensively explored. This can be achieved by the use of antioxidants and/or augmentation of eNOS expression. In 2003 statins were shown to increase the production of endothelial NO in endothelial cell cultures and in animals by the reduction of oxidative stress or by increasing the coupling of the eNOS. It was way back in 1994 that oestrogen was shown to increase both the activity and expression of eNOS. In addition, more recently in 2003, oestrogen was shown to reduce the breakdown of available NO.

Excess of NO: In 2000 it was shown that NO produced from iNOS in vasculature is involved in extensive vasodilatation in septic shock. Later it was demonstrated that inhibition of mitochondrial respiration is an important component of the NO-induced tissue damage. This inhibition of respiration, which is initially NO-dependent and reversible, becomes persistent with time as a result of oxidative stress . Such metabolic hypoxic states where in tissues cannot utilise available oxygen due to NO, could also could also contribute to other inflammatory and degenerative conditions. An obvious therapeutic target for reducing NO production in such conditions would be L-NMMA. L-NMM was tested in a clinical trial for septic shock in 2004. The results were however disappointing probably due to the blanket reduction in NO production from other NOS enzymes there by having deleterious effects on the treatment group. More specific inhibitors for NOS forms are being investigated for in different disease states.

In conclusion, the L-arginine: NO pathway has had a major impact in many areas of research, specially vascular biology. A lot has been understood about this pathway and its interactions, therapeutic targets are being aggressively investigated, but further investigations are required to delineate further the role of NO in human health and disease.

Further reading

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1760731/?tool=pubmed

Nitric Oxide and Platelet Aggregation

Inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production

Nitric Oxide in bone metabolism

Nitric oxide and signalling pathways

Rationale of NO use in hypertension and heart failure

Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function

Nitric oxide: role in Cardiovascular health and disease

Authour/Curator: Aviral Vatsa PhD MBBS

This post is in continuation with posts on basics of NO metabolism and its effect on physiology. Other topics are covered under the following posts.

1. Nitric Oxide and Platelet Aggregation

2. Inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure

3. Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production

4. Nitric Oxide in bone metabolism

5. Nitric oxide and signalling pathways

6. Rationale of NO use in hypertension and heart failure

Nitric oxide plays wide variety of roles in cardiovascular system and acts as a central point for signal transduction pathway in endothelium. NO modulates vascular tone, fibrinolysis, blood pressure and proliferation of vascular smooth muscles. In cardiovascular system disruption of NO pathways or alterations in NO production can result in preponderance to hypertension, hypercholesterolemia, diabetes mellitus, atherosclerosis and thrombosis. The three enzyme isoforms of NO synthase family are responsible for generating NO in different tissues under various circumstances. The endothelial NOS (eNOS) is expressed in endothelial cells, the inducible NOS (iNOS) is expressed in macrophages and neuronal NOS (nNOS) is expressed in certain neurons and skeletal muscle. Although the basic mechanism of action for NO production is the same for all three NOS isoforms, yet deficiencies of each one of them manifest differently or with varying severity in the body e.g. eNOS deficiency might lead to hypertension, more severe form of vascular injury to cerebral ischaemia and more severe form of atherosclerosis induced by hypercholesterolemic diet whereas nNOS deficiency might show less severe form of vascular injury to cerebral ischaemia and absence of iNOS might lead to reduced hypotension in septic shock.

Reduction in NO production is implicated as one of the initial factors in initiating endothelial dysfunction. This reduction could be due to

• reduction in eNOS production

• reduction in eNOS enzymatic activity

• reduced bioavailability of NO

eNOS production is increased by physiological sheer stress on endothelial cells resulting from normal flow of blood along the arterial walls. Alterations in fluid sheer stress patterns e.g due to arterial constriction has been shown to have detrimental effect on eNOS production in endothelial cells. eNOS production is decreased by LDL, angiotensin II and TNF alpha. eNOS is tightly coupled enzyme and its activity can be significantly reduced by reduction in availability of cofactors and substrates, and by competitive inhibitors such as ADMA. Furthermore, uncoupling of eNOS can result in increased production of reactive species of both oxygen (superoxide) and nitrogen (peroxinitrite), which inturn can further reduce eNOS bioavailability. A range of therapeutic targets aim at increasing bioavailability of eNOS and they are summarised here.

Increased production of ROS and peroxinitrite is associated with endothelial dysfunction. Coronary heart disease risk factors may increase NOS mediated ROS formation and peroxinitrite formation. Such risk factors are associated with decreased NO production levels in the vasculature. However, recent data suggests that reduction in bioavailable NO levels in the arteries could be due to increased local oxidative stress rather than reduction in basal NO production.

Oxidation dependent mechanisms have been implicated in endothelial dysfunction. Oxidized low density lipo-proteins (oxLDL) play an important role in early endothelial dysfunction and hence early atherosclerosis (see figure below)

oxLDL can uncouple eNOS and reduced uptake of L-ariginine that can lead to production of superoxide radical oxygen. OxLDL can interfere with NO production and lead to altered NO signalling in the vascular endothelium. In addition, different arteries can be affected differently by these physiological changes e.g. oxLDL affects carotid artery and not the basilar artery thereby implying that intracranial arteries might be protected from endothelium-mediated oxidative injury and hence atherosclerosis. And finally NO can modulate oxidation mediated apoptotic signals in the vessel wall. Hence atherosclerosis can result from the derangement of fine imbalance between NO bioavailability and local oxidative stress.

Therapeutic targets:

There are various pathways being targeted to modulate the bioavailability of eNOS and NO such as

1. Recoupling of eNOS to cofactors and substrates

2. Modulation of eNOS activity by genomic and non genomic mechanisms e.g. by statins, ACE inhibitors, angiotensin II receptor blockers, calcium channel blockers , KLF2 modulators

3. The suppression of inflammatory signalling pathways by PPAR-α activation

4. Modulation of caeviolin mediated endocytosis and thus dissociation of eNOS from caevolin

The above list is not exhaustive, but this post here summarises recent developments in therapeutic targets in NO /eNOS regulation.

Based on:

Nitric Oxide and Pathogenic Mechanisms Involved in the Development of Vascular Diseases

Claudio Napoli and Louis J. Ignarro

Arch Pharm Res Vol 32, No 8, 1103-1108, 2009 , DOI 10.1007/s12272-009-1801-1 

Nitric Oxide Signalling Pathways

Curator/ Author: Aviral Vatsa, PhD, MBBS

In continuation with the previous posts that dealt with short history and chemistry of nitric oxide (NO), here I will try to highlight the pathways involved in NO chemical signalling.

NO is a very small molecule, with a short half life (<5 sec). It diffuses rapidly to its surroundings and is metabolised to nitrites and nitrates. It can travel short distances, a few micrometers, before it is oxidised. Although it was previously believed that NO can only exert its effect for a very short time as other nitrogen oxides were believed to be biologically inert. Recent data suggests that other NO containing compounds such as S- or N-nitrosoproteins and iron-nitrosyl complexes can be reduced back to produce NO. These NO containing compounds can serve as storage and can reach distant tissues via blood circulation, remote from their place of origin. Hence NO can have both paracrine and 'endocrine' effects.

Intracellularly the oxidants present in the cytosol determine the amount of bioacitivity that NO performs. NO can travel roughly 100 microns from NOS enzymes where it is produced. NOS enzymes on the other hand are localised to specific sub-cellular areas, which have relevant proteins in the vicinity as targets for signalling.

NO signalling occurs primarily via three mechanisms (according to Martínez-Ruiz et al):

1. Classical: This occurs via soluble guanylyl cyclase (sGC). Once NO is produced by NOS it diffuses to sGC intracellularly or even in other cells. SGC is highly sensitive for NO, even nanomolar amounts of NO activates sGC, thus making it a potent target for NO in signalling pathways. sGC in turn increases the conversion of GTP to cGMP. cGMP further mediates the regulation of contractile proteins and gene expression pathways via cGMP-activated protein kinases (PKGs). cGMPs cause confirmational changes in PKGs. Signalling by cGMP is terminated by the action of phosphodiestrases (PDEs). PDEs have become major therapeutic targets in the upcoming exciting research projects.
2. Less classical: Within the mitochondria NO can compete with O₂ and inhibit cytochrome c oxidase (CcO) enzyme. This is a reversible inhibition that depends on O₂and NO concentrations and can occur at physiological levels of NO. Various studies have demonstrated that endogenously generated NO can inhibit respiration or that NOS inhibitors can increase respiration at cellular, tissue or whole animal level. Although the exact mechanism of CcO inhibition of NO is still debated, NO-CcO interaction is considered important signalling step in a variety of functions such as inhibition of mitochondrial oxidative phosphorylation, apoptosis and reactive oxygen species (ROS) generation. Interestingly, at higher concentration (~1nM) NO can cause irreversible inhibition of cellular oxidation by reversible and/or irreversible damage to the mitochondrial iron–sulfur centers,In addition to the above mentioned pathways, NO (along with AMP, ROS and O2), can also activate AMP- activated protein kinase (AMPK), an enzyme that plays a central role in regulating intracellular energy metabolism. NO can also regulate hypoxia inducible factor (HIF), an O2-dependent transcription factor that plays a key role in cell adaptation to hypoxia .
3. Non- classical: S-nitrosylation or S-nitrosation is the covalent insertion of NO into thiol groups such as of cysteine residues of proteins. It is precise, reversible, and spatiotemporally restricted post translational modification. This chemical activity is dependent upon the reactivity between nitrosylating agent (a small molecule) and the target (protein residue). It might appear that this generic interaction results in non-specific, wide spread chemical activity with various proteins. However, three factors might determine the regulation of specificity of s-nitrosylation for signalling purposes:

• Subcellular compartmentalisation: high concentrations of nitrosylating agents are required in the vicinity of target residues, thus making it a specific activity.
• Site specificity: certain cysteine residues are more reactive in specific protein microenvironments than others, thus favouring their modification. As a result under physiological conditions only a specific number of cysteine residues would be modified, but under higher NO levels even the slow reacting ones would be modified. Increased impetus in research in this area to determine protein specificity to s-nitrosylation provides huge potential in discovering new therapeutic targets.
• Denitrosylation: different rates of denitrosylation result in s-nitrosylation specificity.

Other modifications in non classical NO mechanisms include S-glutathionylation and tyrosine nitration

Peroxynitrite: It is one of the important reactive nitrogen species that has immense biological relevance. NO reacts with superoxide to form peroxynitrite. Production of peroxynitrite depletes the bioactivty of NO in physiological systems. Peroxynitrite can diffuse through membranes and react with cellular components such as mitochondrial proteins, DNA, lipids, thiols, and amino acid residues. Peroxynitrite can modify proteins such as haemoglobin, myoglobin and cytochrome c. it can alter calcium homeostasis and promote mitochondrial signalling of cell death. However, NO itself in low concentrations have protective action on mitochondrial signalling of cell death.

More details about various aspects of NO signalling can be obtained from the following references.

The post is based on the following Sources:

1. http://www.sciencedirect.com/science/article/pii/S089158491100236X

   http://dx.doi.org/10.1016/j.freeradbiomed.2011.04.010

2. http://content.karger.com/produktedb/produkte.asp?doi=338150

   Cardiology 2012;122:55-68 (DOI: 10.1159/000338150)

3. http://content.onlinejacc.org/article.aspx?articleid=1137266

    J Am Coll Cardiol. 2006;47(3):580-581. doi:10.1016/j.jacc.2005.11.016

4. http://goo.gl/y6oY3

 

In addition, other aspects of NO involvement in biological systems in humans are covered in the following posts on this site:

1. Nitric Oxide and Platelet Aggregation
2. Inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure
3. Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production
4. Nitric Oxide in bone metabolism

Nitric Oxide: Chemistry and function

Curator/Author: Aviral Vatsa PhD, MBBS

Nitric oxide is one of the smallest molecules involved in physiological functions in the body. It is a diatom and thus seeks formation of chemical bonds with its targets rather than structure-function configuration of say protein receptors. Nitric oxide can exert its effects principally by two ways:

• Direct
• Indirect

Direct actions, as the name suggests, result from direct chemical interaction of NO with its targets e.g. with metal complexes, radical species. These actions occur at relatively low NO concentrations (<200 nM)

Indirect actions result from the effects of reactive nitrogen species (RNS) such as NO2 and N2O3. These reactive species are formed by the interaction of NO with superoxide or molecular oxygen. RNS are generally formed at relatively high NO concentrations (>400 nM)

Credits: Nitric Oxide: Biology and Pathobiology By Louis J. Ignarro

Credits: Nitric Oxide: Biology and Pathobiology By Louis J. Ignarro

Although it can be tempting for scientists to believe that RNS will always have deleterious effects and NO will have anabolic effects, this is not entirely true as certain RNS mediated actions mediate important signalling steps e.g. thiol oxidation and nitrosation of proteins mediate cell proliferation and survival, and apoptosis respectively. As depicted in the figure above, NO concentration determines the action it exerts on different proteins. This is highlighted in the following examples from different studies:

• Cells subjected to NO concentration between 10-30 nM were associated with cGMP dependent phosphorylation of ERK
• Cells subjected to NO concentration between 30-60 nM were associated with Akt phosphorylation
• Concentration nearing 100 nM resulted in stabilisation of hypoxia inducible factor-1
• At nearly 400 nM NO, p53 can be modulated
• >1μM NO, it nhibits mitochondrial respiration

Besides the concentration, duration of NO exposure also determines how proteins respond to NO. Hence proteins can be 'immediate' responders or 'delayed' responders. The response can be either 'transient' (short lived) or 'sustained' (prolonged). Different proteins fall into these different categories. These are not rigid categories rather a functional 'classification'.

Endogenously generated NO concentration ranges from 2 nM as in endothelial cell to >1 μM in a fully activated macrophage. This wide range, along with the unique chemical reactivity of NO offers immense versatility to the physiological effects that it can exert in different cellular milieu in the body.

In addition to the concentration-dependent effects, other factors that determine the local cellular/tissue milieu add to the complexities involved with signal transduction undertaken by NO. These factors are

• rate of NO production
• diffusion distance
• rates of consumption
• reactivity of RNS with molecular targets.

These kinetic determinants play vital role in physiological functions and disease states.

Although it is not possible to detail the modes of modulation of biological functions by NO in a short post, but I hope the post gives a taste of the intricacies involved with NO functions and that there are various parameters that determine the exact role of NO in a biological milieu.

Sources

http://www.pnas.org/content/101/24/8894.short

http://onlinelibrary.wiley.com/doi/10.1002/ijc.22336/full

http://cancerres.aacrjournals.org/content/67/1/289.short

http://www.sciencedirect.com/science/article/pii/S0005272806000417

http://goo.gl/eVXFh

Nitric Oxide in bone metabolism

Author: Aviral Vatsa

Nitric oxide (NO) is a short-lived, highly reactive, free radical which is ubiquitously present in the human body. Physiologically, it is widely used as a second messenger both an inter-cellular and intra-cellular signaling molecule. NO is produced when L-arginine is converted to L-citruline in the presence of NO synthase (NOS) enzyme, molecular oxygen, NADPH, and other cofactors. Principally, three isoenzymes of NOS are present in the body to catalyse the production of NO in various anatomic locations and under various physiological conditions. Three distinct genes encode for the three types of NOS i.e. endothelial (eNOS or NOS-3), neuronal (nNOS or NOS-1), and inducible (iNOS or NOS-2) NOS. Neuronal NOS and endothelial NOS are calcium-dependent enzymes, whereas inducible NOS is a calcium-independent inducible enzyme, that is activated and upregulated by cytokines during inflammatory processes. The tissue-specificity indicated in the names is not absolute as these subtypes have been discovered in wider locations in the body.

In bone, NO plays a vital role in mechanosensation and mechanotransduction. Osteocytes are widely accepted as the ‘professional’ mechanosensors in bone. They sense external mechanical loads on bone and produce chemical signals such as NO and prostaglandins. NO in turn has been shown to modulate the activity of both bone forming osteoblasts and bone resorbing osteoclasts. NO is essential for load-induced bone formation in vivo. Studies using single gene deletions have shown that NO is an important cog in the wheel for bone metabolism and bone remodelling. Although eNOS isotype is widely implicated in NO production in bone, but recent studies indicate that iNOS isotype might also be involved in NO production in bone in response to mechanical loading. Targeted deletion of eNOS shows mild osteoporotic phenotype in mice and iNOS pathway has been implicated in L-1-induced osteoclastic bone resorption. Hence both NOS isoforms have important role in bone remodelling.

Challenges to study NO: NO is a small, short-lived signalling molecule. It has a half life of less than 5 sec, which makes its online detection very difficult. Predominantly, the more stable metabolites of NO such as nitrites and nitrates are detected by using techniques such as Greiss reagent. They are however lited by the sensitivity levels and their inability to detect actual levels of NO. However, fluorescent dyes such as DAR 4M and DAF dyes are potent tools to detect online NO production at single cell level. These dyes are membrane-permeable, hence are taken up readily by the cells. Once inside the cell they are metabolised and rendered membrane-impermeable. When cell produces NO these dyes trap NO and get converted into fluorescent product, which can then be detected by using fluorescence microscopy. Moreover, by using these techniques, quantitative analyses of NO production (not only its metabolites) is feasible in live, single cells.

Molecular methods to investigate mRNA or protein levels of NOS enzymes are also used to corroborate with the changes in NO production levels.

Sources:

http://onlinelibrary.wiley.com/doi/10.1359/jbmr.060720/full

http://www.sciencedirect.com/science/article/pii/S0021929007000826

http://www.sciencedirect.com/science/article/pii/S8756328204004144

http://onlinelibrary.wiley.com/doi/10.1359/jbmr.080107/full