Regeneration enhanced in critical-sized bone defects using bone-specific extracellular matrix protein.

Extracellular matrix (ECM) products have the potential to improve cellular attachment and promote tissue-specific development by mimicking the native cellular niche. In this study, the therapeutic efficacy of an ECM substratum produced by bone marrow stem cells (BM-MSCs) to promote bone regeneration in vitro and in vivo were evaluated. Fluorescence-activated cell sorting analysis and phenotypic expression were employed to characterize the in vitro BM-MSC response to bone marrow specific ECM (BM-ECM). BM-ECM encouraged cell proliferation and stemness maintenance. The efficacy of BM-ECM as an adjuvant in promoting bone regeneration was evaluated in an orthotopic, segmental critical-sized bone defect in the rat femur over 8 weeks. The groups evaluated were either untreated (negative control); packed with calcium phosphate granules or granules+BM-ECM free protein and stabilized by collagenous membrane. Bone regeneration in vivo was analyzed using microcomputed tomography and histology. in vivo results demonstrated improvements in mineralization, osteogenesis, and tissue infiltration (114 ± 15% increase) in the BM-ECM complex group from 4 to 8 weeks compared to mineral granules only (45 ± 21% increase). Histological observations suggested direct apposition of early bone after 4 weeks and mineral consolidation after 8 weeks implantation for the group supplemented with BM-ECM. Significant osteoid formation and greater functional bone formation (polar moment of inertia was 71 ± 0.2 mm4 with BM-ECM supplementation compared to 48 ± 0.2 mm4 in untreated defects) validated in vivo indicated support of osteoconductivity and increased defect site cellularity. In conclusion, these results suggest that BM-ECM free protein is potentially a therapeutic supplement for stemness maintenance and sustaining osteogenesis.

The Mouse Heart Attack Research Tool 1.0 database.

The generation of big data has enabled systems-level dissections into the mechanisms of cardiovascular pathology. Integration of genetic, proteomic, and pathophysiological variables across platforms and laboratories fosters discoveries through multidisciplinary investigations and minimizes unnecessary redundancy in research efforts. The Mouse Heart Attack Research Tool (mHART) consolidates a large data set of over 10 yr of experiments from a single laboratory for cardiovascular investigators to generate novel hypotheses and identify new predictive markers of progressive left ventricular remodeling after myocardial infarction (MI) in mice. We designed the mHART REDCap database using our own data to integrate cardiovascular community participation. We generated physiological, biochemical, cellular, and proteomic outputs from plasma and left ventricles obtained from post-MI and no-MI (naïve) control groups. We included both male and female mice ranging in age from 3 to 36 mo old. After variable collection, data underwent quality assessment for data curation (e.g., eliminate technical errors, check for completeness, remove duplicates, and define terms). Currently, mHART 1.0 contains >888,000 data points and includes results from >2,100 unique mice. Database performance was tested, and an example is provided to illustrate database utility. This report explains how the first version of the mHART database was established and provides researchers with a standard framework to aid in the integration of their data into our database or in the development of a similar database. NEW & NOTEWORTHY The Mouse Heart Attack Research Tool combines >888,000 cardiovascular data points from >2,100 mice. We provide this large data set as a REDCap database to generate novel hypotheses and identify new predictive markers of adverse left ventricular remodeling following myocardial infarction in mice and provide examples of use. The Mouse Heart Attack Research Tool is the first database of this size that integrates data sets across platforms that include genomic, proteomic, histological, and physiological data.

A Novel Collagen Matricryptin Reduces Left Ventricular Dilation Post-Myocardial Infarction by Promoting Scar Formation and Angiogenesis.

BACKGROUND: Proteolytically released extracellular matrix (ECM) fragments, matricryptins, are biologically active and play important roles in wound healing. Following myocardial infarction (MI), collagen I, a major component of cardiac ECM, is cleaved by matrix metalloproteinases (MMPs). OBJECTIVES: This study identified novel collagen-derived matricryptins generated post-MI that mediate remodeling of the left ventricle (LV). METHODS: Recombinant collagen Ia1 was used in MMPs cleavage assays, the products were analyzed by mass spectrometry for identification of cleavage sites. C57BL6/J mice were given MI and animals were treated either with vehicle control or p1158/59 matricryptin. Seven days post-MI, LV function and parameters of LV remodeling were measured. Levels of p1158/59 were also measured in plasma of MI patients and healthy controls. RESULTS: In situ, MMP-2 and -9 generate a collagen Iα1 C-1158/59 fragment, and MMP-9 can further degrade it. The C-1158/59 fragment was identified post-MI, both in human plasma and mouse LV, at levels that inversely correlated to MMP-9 levels. We synthesized a peptide beginning at the cleavage site (p1158/59, amino acids 1159 to 1173) to investigate its biological functions. In vitro, p1158/59 stimulated fibroblast wound healing and robustly promoted angiogenesis. In vivo, early post-MI treatment with p1158/59 reduced LV dilation at day 7 post-MI by preserving LV structure (p < 0.05 vs. control). The p1158/59 stimulated both in vitro and in vivo wound healing by enhancing basement membrane proteins, granulation tissue components, and angiogenic factors. CONCLUSIONS: Collagen Iα1 matricryptin p1158/59 facilitates LV remodeling post-MI by regulating scar formation through targeted ECM generation and stimulation of angiogenesis.

Stem cell mechanisms during left ventricular remodeling post-myocardial infarction: Repair and regeneration.

Post-myocardial infarction (MI), the left ventricle (LV) undergoes a series of events collectively referred to as remodeling. As a result, damaged myocardium is replaced with fibrotic tissue consequently leading to contractile dysfunction and ultimately heart failure. LV remodeling post-MI includes inflammatory, fibrotic, and neovascularization responses that involve regulated cell recruitment and function. Stem cells (SCs) have been transplanted post-MI for treatment of LV remodeling and shown to improve LV function by reduction in scar tissue formation in humans and animal models of MI. The promising results obtained from the application of SCs post-MI have sparked a massive effort to identify the optimal SC for regeneration of cardiomyocytes and the paradigm for clinical applications. Although SC transplantations are generally associated with new tissue formation, SCs also secrete cytokines, chemokines and growth factors that robustly regulate cell behavior in a paracrine fashion during the remodeling process. In this review, the different types of SCs used for cardiomyogenesis, markers of differentiation, paracrine factor secretion, and strategies for cell recruitment and delivery are addressed.

Aliskiren and valsartan mediate left ventricular remodeling post-myocardial infarction in mice through MMP-9 effects.

We evaluated whether aliskiren, valsartan, or a combination of both was protective following myocardial infarction (MI) through effects on matrix metalloproteinase (MMP)-9. C57BL/6J wild type (WT, n=94) and MMP-9 null (null, n=85) mice were divided into 4 groups at 3h post-MI: saline (S), aliskiren (A; 50mg/kg/day), valsartan (V; 40mg/kg/day), or A+V and compared to no MI controls at 28days post-MI. All groups had similar infarct areas, and survival rates were higher in the null mice. The treatments influenced systolic function and hypertrophy index, as well as extracellular matrix (ECM) and inflammatory genes in the remote region, indicating that primary effects were on the viable myocardium. Saline treated WT mice showed increased end systolic and diastolic volumes and hypertrophy index, along with reduced ejection fraction. MMP-9 deletion improved LV function post-MI. Aliskiren attenuated the increase in end systolic volume and hypertrophy index, while valsartan improved end diastolic volumes and aliskiren+valsartan improved the hypertrophy index only when MMP-9 was absent. Extracellular matrix and inflammatory gene expression showed distinct patterns among the treatment groups, indicating a divergence in mechanisms of remodeling. This study shows that MMP-9 regulates aliskiren and valsartan effects in mice. These results in mice provide mechanistic insight to help translate these findings to post-MI patients.

Cardiac wound healing post-myocardial infarction: a novel method to target extracellular matrix remodeling in the left ventricle.

Myocardial infarction (MI) is a leading cause of death worldwide. Permanent ligation of the left anterior descending coronary artery (LAD) is a commonly used surgical model to study post-MI effects in mice. LAD occlusion induces a robust wound healing response that includes extracellular matrix (ECM) remodeling. This chapter provides a detailed guide on the surgical procedure to permanently ligate the LAD. Additionally, we describe a prototype method to enrich cardiac tissue for ECM, which allows one to focus on ECM remodeling in the left ventricle following surgically induced MI in mice.

Transgenic overexpression of matrix metalloproteinase-9 in macrophages attenuates the inflammatory response and improves left ventricular function post-myocardial infarction.

Following myocardial infarction (MI), activated macrophages infiltrate into the necrotic myocardium as part of a robust pro-inflammatory response and secrete matrix metalloproteinase-9 (MMP-9). Macrophage activation, in turn, modulates the fibrotic response, in part by stimulating fibroblast extracellular matrix (ECM) synthesis. We hypothesized that overexpression of human MMP-9 in mouse macrophages would amplify the inflammatory and fibrotic responses to exacerbate left ventricular dysfunction. Unexpectedly, at day 5 post-MI, ejection fraction was improved in transgenic (TG) mice (25±2%) compared to the wild type (WT) mice (18±2%; p<0.05). By gene expression profiling, 23 of 84 inflammatory genes were decreased in the left ventricle infarct (LVI) region from the TG compared to WT mice (all p<0.05). Concomitantly, TG macrophages isolated from the LVI, as well as TG peritoneal macrophages stimulated with LPS, showed decreased inflammatory marker expression compared to WT macrophages. In agreement with attenuated inflammation, only 7 of 84 cell adhesion and ECM genes were increased in the TG LVI compared to WT LVI, while 43 genes were decreased (all p<0.05). These results reveal a novel role for macrophage-derived MMP-9 in blunting the inflammatory response and limiting ECM synthesis to improve left ventricular function post-MI.

Matrix metalloproteinase-9 deletion attenuates myocardial fibrosis and diastolic dysfunction in ageing mice.

AIMS: Age-related diastolic dysfunction has been attributed to an increased passive stiffness, which is regulated by extracellular matrix (ECM). We recently showed that matrix metalloproteinase (MMP)-9, an ECM mediator, increases in the left ventricle (LV) with age. The aim of this study, accordingly, was to determine the role of MMP-9 in cardiac ageing. METHODS AND RESULTS: We compared LV function in young (6-9 months), middle-aged (12-15 months), old (18-24 months) and senescent (26-34 months) wild-type (WT) and MMP-9 null mice (n ≥ 12/group). All groups had similar fractional shortenings and aortic peak velocities, indicating that systolic function was not altered by ageing or MMP-9 deletion. The mitral ratios of early to late diastolic filling velocities were reduced in old and senescent WT compared with young controls, and this reduction was attenuated in MMP-9 null mice. Concomitantly, the increase in LV collagen content was reduced in MMP-9 null mice (n = 5-6/group). To dissect the mechanisms of these changes, we evaluated the mRNA expression levels of 84 ECM and adhesion molecules by real-time qPCR (n = 6/group). The expression of pro-fibrotic periostin and connective tissue growth factor (CTGF) increased with senescence, as did transforming growth factor-ß (TGF-ß)-induced protein levels and Smad signalling, and these increases were blunted by MMP-9 deletion. In senescence, MMP-9 deletion also resulted in a compensatory increase in MMP-8. CONCLUSION: MMP-9 deletion attenuates the age-related decline in diastolic function, in part by reducing TGF-ß signalling-induced periostin and CTGF expression and increasing MMP-8 expression to regulate myocardial collagen turnover and deposition.

Temporal and spatial expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases following myocardial infarction.

Following a myocardial infarction (MI), the homeostatic balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) is disrupted as part of the left ventricle (LV) response to injury. The full complement of responses to MI has been termed LV remodeling and includes changes in LV size, shape and function. The following events encompass the LV response to MI: (1) inflammation and LV wall thinning and dilation, (2) infarct expansion and necrotic myocyte resorption, (3) accumulation of fibroblasts and scar formation, and (4) endothelial cell activation and neovascularization. In this review, we will summarize MMP and TIMP roles during these events, focusing on the spatiotemporal localization and MMP and TIMP effects on cellular and tissue-level responses. We will review MMP and TIMP structure and function, and discuss specific MMP roles during both the acute and chronic phases post-MI, which may provide insight into novel therapeutic targets to limit adverse remodeling in the MI setting.

SPARC mediates early extracellular matrix remodeling following myocardial infarction.

Secreted protein, acidic, and rich in cysteine (SPARC) is a matricellular protein that functions in the extracellular processing of newly synthesized collagen. Collagen deposition to form a scar is a key event following a myocardial infarction (MI). Because the roles of SPARC in the early post-MI setting have not been defined, we examined age-matched wild-type (WT; n=22) and SPARC-deficient (null; n=25) mice at day 3 post-MI. Day 0 WT (n=28) and null (n=20) mice served as controls. Infarct size was 52 ± 2% for WT and 47 ± 2% for SPARC null (P=NS), indicating that the MI injury was comparable in the two groups. By echocardiography, WT mice increased end-diastolic volumes from 45 ± 2 to 83 ± 5 µl (P < 0.05). SPARC null mice also increased end-diastolic volumes but to a lesser extent than WT (39 ± 3 to 63 ± 5 µl; P < 0.05 vs. day 0 controls and vs. WT day 3 MI). Ejection fraction fell post-MI in WT mice from 57 ± 2 to 19 ± 1%. The decrease in ejection fraction was attenuated in the absence of SPARC (65 ± 2 to 28 ± 2%). Fibroblasts isolated from SPARC null left ventricle (LV) showed differences in the expression of 22 genes encoding extracellular matrix and adhesion molecule genes, including fibronectin, connective tissue growth factor (CTGF; CCN2), matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of metalloproteinase-2 (TIMP-2). The change in fibroblast gene expression levels was mirrored in tissue protein extracts for fibronectin, CTGF, and MMP-3 but not TIMP-2. Combined, the results of this study indicate that SPARC deletion preserves LV function at day 3 post-MI but may be detrimental for the long-term response due to impaired fibroblast activation.

CC chemokine receptor 5 deletion impairs macrophage activation and induces adverse remodeling following myocardial infarction.

Post-myocardial infarction (MI), chemokine homing of inflammatory cells into the injured left ventricle (LV) regulates ventricular remodeling, in part by stimulating the extracellular matrix response. The CC chemokine receptor 5 (CCR5) is a key chemokine receptor expressed on macrophages, and CCR5 ligands are highly upregulated post-MI. We hypothesized that deletion of CCR5 would attenuate adverse remodeling by decreasing inflammatory cell recruitment. Accordingly, we examined LV function, macrophage recruitment and activation, and collagen content in wild-type (WT, n = 25) and CCR5 null (n = 33) mice at 7 days post-MI. Both groups had similar infarct sizes (44 ± 2% in WT and 42 ± 2% in CCR5 null; P = 0.37). However, the LV remodeling index (end diastolic volume/LV mass) increased to a larger extent in CCR5 null (1.28 ± 0.08 µl/mg for CCR5 null and 1.02 ± 0.06 µl/mg for WT; P < 0.05). Although numbers of infiltrated macrophages were similar in WT and CCR5 null mice, CCR5-deficient macrophages isolated from the infarct zone displayed >50% decrease in gene expression levels of proinflammatory activation markers (interleukin-1ß, interleukin-6, and tumor necrosis factor-α), as well as anti-inflammatory activation markers (arginase 1, CD163, mannose receptor, and transforming growth factor-ß1) compared with WT (all P < 0.05). Concomitant with the reduced macrophage activation, heat shock protein-47 and collagen type I precursor levels in the infarct region decreased in the CCR5 null (1.2 ± 0.3 units in the CCR5 null and 2.3 ± 0.4 units in the WT; P < 0.05), while collagen fragments increased (88.3 ± 5.9 units in the CCR5 null and 32.7 ± 8.5 units in the WT; P < 0.05). We conclude that CCR5 deletion impairs LV remodeling by hindering macrophage activation, which stimulates an imbalance in collagen metabolism and increases the remodeling index.

Proteomic analysis identifies in vivo candidate matrix metalloproteinase-9 substrates in the left ventricle post-myocardial infarction.

Matrix metalloproteinase-9 (MMP-9) deletion has been shown to improve remodeling of the left ventricle post-myocardial infarction (MI), but the mechanisms to explain this improvement have not been fully elucidated. MMP-9 has a broad range of in vitro substrates, but relevant in vivo substrates are incompletely defined. Accordingly, we evaluated the infarct regions of wild-type (wt) and MMP-9 null (null) mice using a proteomic strategy. Wt and null groups showed similar infarct sizes (48+/-3 in wt and 45+/-3% in null), indicating that both groups received an equal injury stimulus. Left ventricle infarct tissue was homogenized and analyzed by 2-DE and MS. Of 31 spot intensity differences, the intensities of 9 spots were higher and 22 spots were lower in null mice compared to wt (all p<0.05). Several extracellular matrix proteins were identified in these spots by MS, including fibronectin, tenascin-C, thrombospondin-1, and laminin. Fibronectin was observed on the gels at a lower than expected molecular weight in the wt group, which suggested substrate cleavage, and the lower molecular weight spot was observed at lower intensity in the MMP-9 null group, which suggested cleavage by MMP-9. Immunoblotting confirmed the presence of fibronectin cleavage products in the wt samples and lower levels in the absence of MMP-9. In conclusion, examining infarct tissue from wt and MMP-9 null mice by proteomic analysis provides a powerful and unique method to identify in vivo candidate MMP substrates.

In vivo matrix metalloproteinase-7 substrates identified in the left ventricle post-myocardial infarction using proteomics.

Matrix metalloproteinase-7 (MMP-7) deletion has been shown to improve survival after myocardial infarction (MI). MMP-7 has a large array of in vitro substrates, but in vivo substrates for MMP-7 following MI have not been fully identified. Accordingly, we evaluated the infarct regions of wild-type (WT; n = 12) and MMP-7 null (null; n = 10) mice using a proteomic strategy. Seven days post-MI, infarct regions of the left ventricles were excised, homogenized, and protein extracts were analyzed by two-dimensional gel electrophoresis and mass spectrometry. Of 13 spots that showed intensity differences between WT and null, the intensities of eight spots were higher and those of five spots were lower in the null group (p < 0.05). Fibronectin and tenascin-C, known in vitro substrates of MMP-7, were identified in spots that showed lower intensity in the null. Immunoblotting and in vitro cleavage assays confirmed reduced fibronectin and tenascin-C fragment generation in the null, and this effect was restored by exogenous administration of MMP-7. Lower levels of full-length peroxiredoxin-1 and -2 and higher levels of the full-length peroxiredoxin-3 were detected in the null group, suggesting MMP-7 deletion may also indirectly regulate protein levels through nonenzymatic mechanisms. In conclusion, this is the first study to identify fibronectin and tenascin-C as in vivo MMP-7 substrates in the infarcted left ventricle using a proteomic approach.

Extracellular matrix turnover and signaling during cardiac remodeling following MI: causes and consequences.

The concept that extracellular matrix (ECM) turnover occurs during cardiac remodeling is a well-accepted paradigm. To date, a multitude of studies document that remodeling is accompanied by increases in the synthesis and deposition of ECM components as well as increases in extracellular proteases, especially matrix metalloproteinases (MMPs), which break down ECM components. Further, soluble ECM fragments generated from enzymatic action serve to stimulate cell behavior and have been proposed as candidate plasma biomarkers of cardiac remodeling. This review briefly summarizes our current knowledge base on cardiac ECM turnover following myocardial infarction (MI), but more importantly extends discussion by defining avenues that remain to be explored to drive the ECM remodeling field forward. Specifically, this review will discuss cause and effect roles for the ECM changes observed following MI and the potential role of the ECM changes that may serve as trigger points to regulate remodeling. While the pattern of remodeling following MI is qualitatively similar but quantitatively different from various types of injury, the basic theme in remodeling is repeated. Therefore, while we use the MI model as the prototype injury model, the themes discussed here are also relevant to cardiac remodeling due to other types of injury.

C-terminal fragment of transforming growth factor beta-induced protein (TGFBIp) is required for apoptosis in human osteosarcoma cells.

Transforming growth factor beta-induced protein (TGFBIp), is secreted into the extracellular space. When fragmentation of C-terminal portions is blocked, apoptosis is low, even when the protein is overexpressed. If fragmentation occurs, apoptosis is observed. Whether full-length TGFBIp or integrin-binding fragments released from its C-terminus is necessary for apoptosis remains equivocal. More importantly, the exact portion of the C-terminus that conveys the pro-apoptotic property of TGFBIp is uncertain. It is reportedly within the final 166 amino acids. We sought to determine if this property is dependent upon the final 69 amino acids containing the integrin-binding, EPDIM and RGD, sequences. With MG-63 osteosarcoma cells, transforming growth factor (TGF)-beta1 treatment increased expression of TGFBIp over 72 h (p<0.001). At this time point, apoptosis was significantly increased (p<0.001) and was prevented by an anti-TGFBIp, polyclonal antibody (p<0.05). Overexpression of TGFBIp by transient transfection produced a 2-fold increase in apoptosis (p<0.01). Exogenous purified TGFBIp at concentrations of 37-150 nM produced a dose dependent increase in apoptosis (p<0.001). Mass spectrometry analysis of TGFBIp isolated from conditioned medium of cells treated with TGF-beta1 revealed truncated forms of TGFBIp that lacked integrin-binding sequences in the C-terminus. Recombinant TGFBIp truncated, similarly, at amino acid 614 failed to induce apoptosis. A recombinant fragment encoding the final 69 amino acids of the TGFBIp C-terminus produced significant apoptosis. This apoptosis level was comparable to that induced by TGF-beta1 upregulation of endogenous TGFBIp. Mutation of the integrin-binding sequence EPDIM, but not RGD, blocked apoptosis (p<0.001). These pro-apoptotic actions are dependent on the C-terminus most likely to interact with integrins.

Prevalence of diabetes mellitus and correlation of urinary transforming growth factor-beta1 with blood hemoglobin A1C in the Atascosa Diabetes Study.

INTRODUCTION: This study was conducted to determine the prevalence of type 2 diabetes and prediabetes in the Atascosa Diabetes Study sample and to ascertain the relationship between urinary transforming growth factor-beta1 (TGF-beta1) and blood hemoglobin (Hgb) A1C. METHODS: Subjects (N = 526) classified as adjusted normal, at risk, prediabetes, and diabetes mellitus were given a one-hour and two-hour postprandial glucose (PPG) test. Morning urine samples were collected to test for a correlation of TGF-beta1 with blood HgbA1C. RESULTS: Of the subjects, 14.3% had diabetes, 31.6% had prediabetes, 7.9% were at risk, and 46.2% were adjusted normal. Sensitivity and specificity for one-hour PPG for prediabetes and diabetes were significant, with an efficiency of 80.2%-90.9% and a likelihood ratio of 4.7-10.2. Receiver operating characteristic analysis resulted in an area under the curve of .880 +/- .016 for one hour to prediabetes and diabetes and .960 +/- .016 for one hour to diabetes. Prediabetes was 1.07 times more prevalent in Hispanics, but diabetes was 1.65 times greater in Whites. Urinary TGF-beta1 was more than fivefold higher in poorly controlled versus controlled diabetic or normal subjects and had a significant positive correlation with HgbA1C. CONCLUSIONS: The percentage of subjects with type 2 diabetes was 1.64 times higher than the national average. Prevalence of prediabetes was equivalent in Hispanics and Whites, and the reversal for diabetes might reflect higher mortality rate from diabetes in Hispanics in Atascosa County. Use of one-hour PPG and urine markers for early kidney involvement could improve this disparity in such high-risk populations.

Numerical modeling of ferrous-ion oxidation rate in Acidithiobacillus ferrooxidans ATCC 23270: optimization of culture conditions through statistically designed experiments.

Statistically designed experimental strategy has been performed in order to evaluate and optimize nutritional and environmental parameters that affect ferrous ion oxidation rate in Acidithiobacillus ferrooxidans ATCC 23270. Plackett-Burman design was carried out to evaluate efficiently the biological significance of 10 culture conditions influencing ferrous-ion oxidation rate of A. ferrooxidans grown for 5 days in shake-flask batch mode on the newly modified 9-K media. Among ten fermentation factors examined, the most significant variables influencing ferrous-ion oxidation rate were statistically elucidated to be pH and calcium nitrate as positive contributors, whereas trace metals solution and potassium chloride were the most significant negative contributors. The optimal levels of the most significant three nutritional factors were further predicted from a polynomial model created from the data obtained from three level factorial design, a Box-Behnken design. Predicted optimal ferrous-ion oxidation rate Q(Fe2+) was recorded to be 0.148 (g Fe2+/l/hr). On verifying the predicted value, an experiment was performed under optimal predicted conditions and showed an actual experimental Q(Fe2+) of 0.152 g/l/hr, which was 2.7% over the predicted value. Our optimized medium formula gave overall five folds increase in ferrous-ion oxidation rates over the previously published data of standard 9-K medium on batch culture of A. ferrooxidans ATCC 23270 with higher mu(max) (hr(-1)) of 0.177 which was achieved within 75 h incubation in shake-flask culture.