Millán Lab Research
Our Research on ‘Mechanisms of Skeletal Mineralization’
The Millán laboratory works on understanding the mechanisms controlling normal and pathological mineralization. These studies use knockout, knockin and transgenic mouse models to examine the function of the tissue-nonspecific alkaline phosphatase isozyme (TNAP), the nucleosidetriphosphate pyrophosphohydrolase-1 (NPP1), Ankylosis (ANK) protein and osteopontin (OPN), all molecules that act in concert to regulate the extracellular concentrations of inorganic pyrophosphate (PPi), a potent inhibitor of mineralization (Hessle et al., 2002; Harmey et al., 2004). In turn, extracellular PPi (ePPi) concentrations regulate the expression of OPN, another important regulator of mineralization. Current work focuses on testing the central hypothesis that the ePPi-mediated regulation of OPN and the OPN-mediated regulation of ePPi are linked counter-regulatory mechanisms that control the concentrations of these two important mineralization inhibitors, OPN and ePPi. In addition we study the mechanisms that control the initiation of bone mineral (hydroxyapatite) formation inside osteoblast- and chondrocytes-derived matrix vesicles (Ciancaglini et al., 2010) and the non-redundant role of TNAP and PHOSPHO1 in this process. Double ablation of PHOSPHO1 and TNAP function in mice leads to the complete absence of skeletal mineralization and perinatal lethality (Yadav et al., 2010). The current comprehensive model that guides our work postulates intravesicular PHOSPHO1 function and Pi influx into matrix vesicles in the initiation of mineralization and the functions of TNAP, NPP1 and collagen in the extravesicular progression of skeletal mineralization. These basic mechanistic studies are guiding the development of novel therapeutic strategies for hypophosphatasia and for medial vascular calcification, and may also be of relevance for osteoarthritis and osteoporosis.
Enzyme Replacement Therapy for ‘Hypophosphatasia’
“Hypophosphatasia” (HPP) is an inborn-error-of-metabolism characterized by low serum alkaline phosphatase activity caused by loss-of-function mutations within the gene that encodes TNAP. Consequently, ePPi, a natural substrate of TNAP, accumulates endogenously in HPP blocking hydroxyapatite crystal growth within the skeletal matrix leading to the characteristic rickets (soft bones in children) or osteomalacia (soft bones in adults). The clinical severity of HPP ranges widely and spans complete absence of bone mineralization and stillbirth, to spontaneous fractures and loss of teeth in adult life. In perinatal HPP, profound skeletal disease is obvious at birth. Prolonged survival is rare. Infantile HPP presents before six months-of-age with failure-to-thrive, rickets, and hypotonia and sometimes hypercalcemia, nephrocalcinosis, and epilepsy. The prevalence of severe hypophosphatasia is estimated to be 1:100,000 in a population of largely Anglo Saxon origin but can reach 1:2,500 newborns in Canadian Mennonites. There is no established medical treatment for HPP.
Using a mouse model of infantile HPP developed in our laboratory, we recently succeeded in preventing all the manifestations of HPP by daily subcutaneous injections of a bone-targeted form of TNAP (Millán et al., 2008). This enzyme replacement approach is now being evaluated in clinical trials in infants and children with HPP. For more information on these ongoing trials and the company manufacturing bone-targeted TNAP click here. More recently, we have also succeeded in delivering bone-targeted TNAP by a single intravenous injection at birth of a lentiviral vector harboring the therapeutic gene construct. The HPP mice survived more than a year without evidence of HPP disease (Yamamoto et al., 2010). Current studies underway in the laboratory focus on testing the use of lentiviral vector treatment for HPP in utero as well as assessing the use of adeno-associated viruses as the delivery vectors for HPP. We are also creating and characterizing models of adult and odonto HPP to evaluate their response to enzyme replacement and gene therapy.
Developing Treatments for ‘Medial Vascular Calcification’
During our studies on the mechanisms that control the production and degradation of ePPi, we observed that genetic deficiencies in NPP1 or ANK functions lead to a reduction in systemic ePPi levels and soft-tissue calcification, including vascular calcification (Murshed et al., 2005; Narisawa et al., 2007). We found a simultaneous increase in TNAP activity in the vascular smooth muscle cells of Enpp1 knockout and ank/ank mutant mice, which likely contribute to the decreased output of ePPi by these calcifying cells. Upregulation of TNAP activity was also documented in the aortas of uremic rats, a model of vascular calcification associated with end-stage renal disease (Lomashvili et al., 2008). The finding that TNAP upregulation is a common denominator at sites of medial calcification has important translational implications and has prompted us to undertake the development of potent small molecule inhibitors of TNAP’s pyrophosphatase activity for therapeutic use in the prevention and treatment of medial calcification (Narisawa et al., 2007; Sidique et al., 2009; Dahl et al., 2009; Sergienko et al., 2009; Chung et al., 2010; Sergienko and Millán, 2010). Current efforts are focussed on improving the lead compounds at hand via Medicinal Chemistry efforts to enable proof-of-concept studies in experimental animals towards the goal of developing a treatment for medial vascular calcification.
Understanding ‘Gastrointestinal Physiology’
Another area of interest in the Millán laboratory is gut physiology, inflammation and tumorigenesis and the role of intestinal alkaline phosphatases (IAPs) in these processes. Our studies have shown that the mouse gut expresses three IAP genes, Akp3, Akp5 and Akp6 (Narisawa et al., 2007). Akp3 encodes a duodenal-specific isozyme of IAP clearly involved in fatty acid absorption, since Akp3 knockout mice display an accelerated transport of fatty acids in their gut (Narisawa et al., 2003), become obese and develop hepatic steatosis (fatty liver) when fed a high fat diet (Nakano et al., 2007). Akp3 is also involved in LPS dephosphorylation and plays a fundamental role establishing gut mucosal immunity (Goldberg et al., 2008) and is essential to maintaining a healthy symbiotic environment with our gut bacterial flora. These studies are of relevance to elucidating the pathogenesis of, and developing treatments for, conditions such as inflammatory bowel disease (IBD), Crohn’s Disease (CD), gut mucosal barrier dysfunction during starvation, diarrheal diseases and colon cancer.