Adopting the Rumsfeld approach to understanding the action of levodopa and apomorphine in Parkinson's disease.

Dopaminergic therapies dominate the treatment of the motor and non-motor symptoms of Parkinson's disease (PD) but there have been no major advances in therapy in many decades. Two of the oldest drugs used appear more effective than others-levodopa and apomorphine-but the reasons for this are seldom discussed and this may be one cause for a lack of progress. This short review questions current thinking on drug action and looks at whether adopting the philosophy of ex-US Secretary of State Donald Rumsfeld reveals 'unknown' aspects of the actions of levodopa and apomorphine that provide clues for a way forward. It appears that both levodopa and apomorphine have a more complex pharmacology than classical views would suggest. In addition, there are unexpected facets to the mechanisms through which levodopa acts that are either forgotten as 'known unknowns' or ignored as 'unknown unknowns'. The conclusion reached is that we may not know as much as we think about drug action in PD and there is a case for looking beyond the obvious.

Biological mechanisms of bone and cartilage remodelling--genomic perspective.

Rapid advancements in the field of genomics, enabled by the achievements of the Human Genome Project and the complete decoding of the human genome, have opened an unimaginable set of opportunities for scientists to further unveil delicate mechanisms underlying the functional homeostasis of biological systems. The trend of applying whole-genome analysis techniques has also contributed to a better understanding of physiological and pathological processes involved in homeostasis of bone and cartilage tissues. Gene expression profiling studies have yielded novel insights into the complex interplay of osteoblast and osteoclast regulation, as well as paracrine and endocrine control of bone and cartilage remodelling. Mechanisms of new bone formation responsible for fracture healing and distraction osteogenesis, as well as healing of joint cartilage defects, have also been extensively studied. Microarray experiments have been especially useful in studying pathological processes involved in diseases such as osteoporosis or bone tumours. Existing results show that microarrays hold great promise in areas such as identification of targets for novel therapies or development of new biomarkers and classifiers in skeletal diseases.

BMP-6 exerts its osteoinductive effect through activation of IGF-I and EGF pathways.

We have recently shown that human recombinant BMP-6 (rhBMP-6), given systematically, can restore bone in animal models of osteoporosis. To further elucidate the underlying mechanisms of new bone formation following systemic application of BMPs, we conducted gene expression profiling experiments using bone samples of oophrectomised mice treated with BMP-6. Gene set enrichment analysis revealed enrichment of insulin-like growth factor-I and epidermal growth factor related pathways in animals treated with BMP-6. Significant upregulation of IGF-I and EGF expression in bones of BMP-6 treated mice was confirmed by quantitative PCR. To develop an in vitro model for evaluation of the effects of BMP-6 on cells of human origin, we cultured primary human osteoblasts. Treatment with rhBMP-6 accelerated cell differentiation as indicated by the formation of mineralised nodules by day 18 of culture versus 28-30 days in vehicle treated cultures. In addition, alkaline phosphatase gene expression and activity were dramatically increased upon BMP-6 treatment. Expression of IGF-I and EGF was upregulated in human osteoblast cells treated with BMP-6. These results collectively indicate that BMP-6 exerts its osteoinductive effect, at least in part, through IGF-I and EGF pathways, which can be observed both in a murine model of osteopenia and in human osteoblasts.

Role of EP2 and EP4 receptor-selective agonists of prostaglandin E(2) in acute and chronic kidney failure.

We tested the efficacy of three selective agonists of prostaglandin E(2) (PGE(2)) receptor, EP2 (CP-536,745-01), EP2/4 (CP-043,305-02), and EP4 (CP-044,519-02), in two models of acute and chronic kidney failure. In the nephrotoxic mercury chloride (HgCl(2)) rat model of acute kidney failure systemically administered EP4 agonist reduced the serum creatinine values and increased the survival rate. Although the EP2 or the EP2/4 agonist did not change the serum creatinine values, the EP2 receptor agonist increased the survival rate. Histological evaluation of kidneys from EP4-treated rats indicated less proximal tubular necrosis and less apoptotic cells. In a rat model of chronic renal failure, the three receptor agonists decreased the serum creatinine and increased the glomerular filtration rate at 9 weeks following therapy. Kidneys treated with the EP4 agonist had less glomerular sclerosis, better preservation of proximal and distal tubules and blood vessels, increased convoluted epithelium proliferation and less apoptotic cells. Nephrectomy had no influence on the expression of the EP4 receptor, whereas EP2 receptor expression was reduced by 50% and then corrected following treatment with EP2 and EP2/4 receptor agonists. These findings suggest that PGE(2) has an important role in acute kidney failure via the EP4 receptor, whereas in chronic kidney failure both EP2 and EP4 receptors are equally important in preserving the progression of chronic kidney failure. Thus, agonism of EP2 and EP4 receptors may provide a basis for treating acute and chronic kidney failure.

Genome-wide expression profiling of human blood reveals biomarkers for Huntington's disease.

Huntington's disease (HD) is an autosomal dominant disorder caused by an expansion of glutamine repeats in ubiquitously distributed huntingtin protein. Recent studies have shown that mutant huntingtin interferes with the function of widely expressed transcription factors, suggesting that gene expression may be altered in a variety of tissues in HD, including peripheral blood. Affymetrix and Amersham Biosciences oligonucleotide microarrays were used to analyze global gene expression in blood samples of HD patients and matched controls. We identified 322 mRNAs that showed significantly altered expression in HD blood samples, compared with controls (P < 0.0005), on two different microarray platforms. A subset of up-regulated mRNAs selected from this group was able to distinguish controls, presymptomatic individuals carrying the HD mutation, and symptomatic HD patients. In addition, early presymptomatic subjects showed gene expression profiles similar to those of controls, whereas late presymptomatic subjects showed altered expression that resembled that of symptomatic HD patients. These elevated mRNAs were significantly reduced in HD patients involved in a dose-finding study of the histone deacetylase inhibitor sodium phenylbutyrate. Furthermore, expression of the marker genes was significantly up-regulated in postmortem HD caudate, suggesting that alterations in blood mRNAs may reflect disease mechanisms observed in HD brain. In conclusion, we identified changes in blood mRNAs that clearly distinguish HD patients from controls. These alterations in mRNA expression correlate with disease progression and response to experimental treatment. Such markers may provide clues to the state of HD and may be of predictive value in clinical trials.

Anomalous branch of the left common carotid artery.

A previously unreported anomalous branch of the left common carotid artery (LCCA) was observed during dissection at the Zagreb Medical School. The anomalous branch arose from the anterior surface of the LCCA approximately 2 cm superior to the aortic arch, and subsequently bifurcated into a right and left branch. We describe its anatomic features. Clinical implications are also discussed.

Distraction osteogenesis by Ilizarov and unilateral external fixators in a canine model.

We studied distraction osteogenesis in canine experimental model using two types of external fixators, Ilizarov (n=6) or AO unilateral (n=9) external fixator. Distraction started 1 week after surgery (2 x 0.5 mm/day) and lasted for 3 weeks. Specimens were harvested from weeks 7 through 12. The outcome was assessed by X-ray, histology, histomorphometry and microradiography. Bone regeneration as observed by X-rays was satisfactory and similar in both groups. Both endochondral ossification and intramembranous ossification were found simultaneously in both groups. In both groups, bone formation parameters were significantly higher in the area of consolidating bone. No differences in histomorphometric parameters existed between the groups. In the study period, the bone formation was enhanced and prevailed in the distraction area. This study demonstrated the utility of the canine experimental model for the study of distraction osteogenesis.

Regulation of BMP-7 expression by retinoic acid and prostaglandin E(2).

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) gene superfamily of growth and differentiation factors. Members of the BMP family were originally cloned and characterized by their ability to induce ectopic bone formation. Of the various BMPs cloned, the bone inductive ability of BMP-7 (OP-1) and BMP-2 has been well characterized. Both BMP-7 and -2 have been shown to have clinical utility in the healing of non-union fractures. However, in spite of the various advances in BMP research, the physiological regulation of BMPs is not well understood. Here we studied the expression of BMP-7 by cloning a 4.6-kB fragment of the human BMP-7 promoter (hBMP-7p) and placing it upstream of a luciferase reporter. The promoter reporter construct was stably transfected into different cell backgrounds and its regulation by various factors was investigated. We show that retinoic acid (RA) treatment results in an upregulation of the hBMP-7p reporter activity. This regulation of the hBMP-7p was further confirmed by Northern blot, PCR, and Western blot analyses, which showed an increase in both BMP-7 mRNA and protein expression upon treatment with RA. We further show that RA specifically upregulates expression of osteocalcin via activation of BMP-7 mRNA and protein in vitro. Similarly, prostaglandin E(2) (PGE(2)) treatment increases BMP-7 mRNA and protein levels, but does not transcriptionally activate the hBMP-7p. Additionally, in vivo expression of BMP-7 in bone was increased upon PGE(2) treatment. In conclusion, RA and PGE(2) upregulate BMP-7 protein expression both in vitro and in vivo.

An EP2 receptor-selective prostaglandin E2 agonist induces bone healing.

The morbidity and mortality associated with impaired/delayed fracture healing remain high. Our objective was to identify a small nonpeptidyl molecule with the ability to promote fracture healing and prevent malunions. Prostaglandin E2 (PGE2) causes significant increases in bone mass and bone strength when administered systemically or locally to the skeleton. However, due to side effects, PGE2 is an unacceptable therapeutic option for fracture healing. PGE2 mediates its tissue-specific pharmacological activity via four different G protein-coupled receptor subtypes, EP1, -2, -3, and -4. The anabolic action of PGE2 in bone has been linked to an elevated level of cAMP, thereby implicating the EP2 and/or EP4 receptor subtypes in bone formation. We identified an EP2 selective agonist, CP-533,536, which has the ability to heal canine long bone segmental and fracture model defects without the objectionable side effects of PGE2, suggesting that the EP2 receptor subtype is a major contributor to PGE2's local bone anabolic activity. The potent bone anabolic activity of CP-533,536 offers a therapeutic alternative for the treatment of fractures and bone defects in patients.