Abstract
Basic fibroblast growth factor (bFGF) is a pleiotropic mitogen with a potent bone-forming effect, rendering it a potential osteoporosis therapy. This study examined selected extraskeletal effects of bFGF in ovariectomized rats, a well-established model of human postmenopausal osteopenia, to more fully characterize side effects associated with bFGF treatment. Five-month-old, osteopenic, ovariectomized rats were injected subcutaneously with vehicle or bFGF (1 mg/kg) daily for 3 weeks. Hematologic and biochemical analyses were performed; and kidneys, livers, and proximal tibiae were examined histologically and histomorphometrically. bFGF administration resulted in anemia that was due to a shift toward granulocyte production in the bone marrow. Increased granulocyte production was also observed in the liver of bFGF-treated rats, which exhibited a markedly increased number and area of hematopoietic foci. bFGF administration also caused mild glomerular hypertrophy that was not attended by significant biochemical evidence of glomerular dysfunction. The bone anabolic effect of subcutaneous bFGF administration was confirmed in the proximal tibia, and was associated with a significant decrease in urine fractional excretion of calcium in bFGF-treated rats. Though bFGF strongly stimulates bone formation at osteopenic skeletal sites, its extraskeletal effects may restrict the long-term use of bFGF in its current form as an osteoporosis therapy.
Keywords
Introduction
Basic fibroblast growth factor (bFGF) is a widely distributed, heparin-binding, polypeptide growth factor with mitogenic effects in various cells of mesodermal origin (Gospodarowicz et al., 1987). The ability of bFGF to stimulate the proliferation and differentiation of mesenchymal stem cells and osteoblast-like cells in vitro (Canalis et al., 1988; Globus et al., 1988; Pitaru et al., 1993; Martin et al., 1997) suggested that this growth factor may stimulate bone formation. Subsequent in vivo studies showed that IV treatment with bFGF markedly increased the osteoblast population and augmented bone mass in intact rats (Mayahara et al., 1993; Nagai et al., 1995; Nakamura et al., 1995). These initial findings were confirmed in ovariectomized (OVX) rats, a well-established animal model for postmenopausal bone loss (Kalu, 1991). bFGF was found to have a strong stimulatory effect on bone formation and the ability to increase cancellous bone matrix in osteopenic OVX rats (Liang et al., 1999; Wronski et al., 2001; Iwaniec et al., 2003), which focused attention on the growth factor as a potential osteoporosis therapy. Compared to other bone anabolic agents studied (Zerwekh et al., 1994; Hodsman et al., 1998; Wronski and Li, 1998), bFGF treatment is distinguished by the induction of osteoid formation in the absence of preexisting trabecular template (Iwaniec et al., 2003). Therefore, bFGF demonstrates unique potential among bone anabolic agents as a treatment for postmenopausal osteoporosis, especially for those patients who have inadequately responded to conventional therapeutic options (Wronski, 2001).
Evaluation of a potential treatment for use in humans must involve characterization of its adverse side effects. Reversible, dose-dependent, pathology in tissues and organs other than bone has been observed in association with systemic bFGF administration. Reported side effects include transient hypotension, anemia, thrombocytopenia, and proteinuria in dogs (Lazarous et al., 1995, 1996; Unger et al., 2000) and anemia, hepatic extramedullary hematopoiesis, and renal toxicity in monkeys (Mazu′e et al., 1992, 1993). Rats treated with bFGF have exhibited a lower rate of weight gain, anemia, hypophosphatemia, proteinuria, hypertrophy of glomerular and alveolar epithelia, hepatic extramedullary hematopoiesis, decreased longitudinal bone growth in growing rats, endosteal hyperostosis, and impaired bone calcification (Mazu′e et al., 1992, 1993; Mayahara et al., 1993; Nagai et al., 1995; Liang et al., 1999; Wronski et al., 2001; Iwaniec et al., 2002). These effects were induced by IV treatment with bFGF and, in some cases, were described in a qualitative manner only. A more recent study indicated that SC treatment with a high dose of bFGF, much like the IV route of administration, has a strong bone anabolic effect in OVX rats (Iwaniec et al., 2003). The purpose of the current study was to employ histomorphometry in addition to traditional methods to characterize select extraskeletal side effects of SC bFGF administration in OVX rats with a focus on hematology, serum and urine biochemistry, bone marrow hematopoiesis, extramedullary hematopoiesis, and glomerular morphology.
Materials and Methods
Animals
The experimental animals were female Sprague–Dawley rats (Charles River Laboratory, Wilmington, MA) that were 3 months of age and weighed an average of 228 g at the beginning of the study. The rats were ovariectomized from a paralumbar approach under ketamine (70 mg/kg) and xylazine (10 mg/kg) anesthesia. All animals were housed individually at 21°C with a 12 h/12 h light/dark cycle. The food consumption (Teklad 22/5 Rodent Diet, Madison, WI) for each rat was restricted to approximately 18 g daily to minimize the increase in body weight associated with ovariectomy (Wronski et al., 1987). The care and treatment of the animals was approved by the Institutional Animal Care and Use Committee at the University of Florida (Gainesville, FL).
Treatments
At 2 months postovariectomy, one group of OVX rats (n = 9) was injected SC with phosphate-buffered saline vehicle daily for 3 weeks. The second group of OVX rats (n = 10) was subjected to daily SC injections of bFGF (Chiron Corp., Emeryville, CA) for 3 weeks. The growth factor was dissolved in phosphate-buffered saline and administered at a dose of 1 mg/kg. At the end of the treatment period, all rats were anesthetized with ketamine and xylazine as described above and sacrificed by exsanguination from the abdominal aorta.
Hematology and Biochemistry
The packed red blood cell volume was measured at the time of necropsy with a microhematocrit reader (Clay Adams, Dickinson and Co., Parsippany, NJ). Serum and urine samples were collected for biochemical analyses and stored at −80°C for no more than 3 days prior to analysis. The following analytes were measured using an automated biochemistry analyzer (Hitachi 911, Boehringer Mannheim Corp., Indianapolis, IN): serum albumin (bromcresol green method, Diagnostic Chemicals Limited, Oxford, CT), urine microprotein (pyrogallol red with molybdenum acid method, Diagnostic Chemicals Limited, Oxford, CT), serum and urine creatinine (modified Jaffe reaction, Diagnostic Chemicals Limited, Oxford, CT), serum and urine calcium (o-cresolphthalein complexone method, Thermo Electron Corp., Louisville, CO), and serum and urine phosphorus (phosphomolybdate method, Diagnostic Chemicals Limited, Oxford, CT). Data obtained from these measurements were used to calculate the urine protein-to-creatinine ratio, the urine fractional excretion of calcium, and the urine fractional excretion of phosphorus. Fractional excretions were calculated with the formula: fractional excretion of a substance = (Ux/Sx) ÷ (UCr/SCr); where Ux is the urine concentration of the substance, Sx is the serum concentration of the substance, UCr is the urine concentration of creatinine, and SCr is the serum concentration of creatinine. Serum and urine specimens were not available for all animals in the study, because, a low number of rats died under deep sedation, before blood could be collected for serum measurements, and a low number of rats did not have urine within the urinary bladder at the time of euthanasia. For serum biochemistry in vehicle-treated animals, the n was 8; and for urine biochemistry, the n was 7. For serum biochemistry in bFGF-treated animals, the n was 8; for urine protein-to-creatinine ratio calculation, the n was 7; and for fractional excretion calculations, the n was 6.
Histology and Histomorphometry
Tissue Preparation
The right proximal tibia, a portion of the left medial lobe of the liver, and half of the left kidney were placed in 10% phosphate-buffered formalin for tissue fixation. Subsequently, the tissues were dehydrated in graded alcohols and xylene, embedded in modified methyl methacrylate, and sectioned at 3 to 5 μm thickness with Leica/Jung 2065 and 2165 microtomes. The sections were adhered to 1% gelatin-coated glass microscope slides, deplasticized in methoxyethylacetate, and stained with Harris’ hematoxylin and eosin Y with phloxine B counterstain (Fisher Scientific, Pittsburgh, PA). For analysis of the osteogenic skeletal effects of bFGF, sections from the proximal tibiae were stained according to the von Kossa method with a tetrachrome counter-stain (Polysciences, Warrington, PA) as previously described for the lumbar vertebral bodies from these animals (Iwaniec et al., 2003).
Evaluation of Osteogenesis
Two nonconsecutive sections per animal were used to measure osteoblast surface, osteoid surface, and osteoclast surface in the secondary spongiosa of the proximal tibial metaphysis (Liang et al., 1999). The measurements were performed at 200× magnification with the Bioquant Bone Morphometry System (R&M Biometrics Corp., Nashville, TN).
Evaluation of Bone Marrow Hematopoiesis
The bone marrow of each proximal tibia was examined by routine light microscopy and a 500 cell differential count was performed to determine the number of leukocytes with granulocytic (myeloid) differentiation and the number of nonleukocytes with erythroid differentiation. Using these quantities, the granulocytic-to-erythroid (G:E) ratio was calculated. The medullary hematopoietic area of the proximal tibia was measured histomorphometrically at 200 × magnification using the OsteoMeasure system (OsteoMetrics, Inc., Atlanta, GA) (Figure 1 A, B). A total area of 3.33 mm2 immediately distal to the proximal growth plate was evaluated in each tibia. Regions of the marrow that contained hematopoietic cells were manually demarcated using OsteoMeasure. The demarcated regions represented the hematopoietic area, which was used to calculate the percent hematopoietic area per marrow area for each section.
Evaluation of Extramedullary Hematopoiesis
Two nonconsecutive sections of liver per animal were examined histomorphometrically at 100 × magnification in a manner similar to that performed in the bone marrow. Eighteen consecutive fields comprising a total area of 4 mm2 per section (8 mm2 per animal) were examined. Foci of extramedullary hematopoiesis were counted and manually demarcated using OsteoMeasure. The demarcated regions represented the extramedullary hematopoietic area, which was used to calculate the percent hematopoietic area per hepatic tissue area for each section.
Evaluation of Glomerular Size
Two nonconsecutive sections of kidney per animal were examined histomorphometrically at 200× magnification. To minimize imprecision in glomerular measurements due to variable bisection of spherical structures, the renal sections were initially scanned to identify the 10 largest glomerular cross sections per slide (20 glomeruli per animal). Measurements of the glomerular diameter and the thickness of Bowman’s capsule parietal epithelium were performed on these 10 glomeruli (Figure 1C and D). To minimize inaccuracy in glomerular measurements due to variable round-to-oval shape, each glomerulus was manually labeled using OsteoMeasure with 4 to 6 radial bisections that crossed through the center of the glomerulus. The average length of these bisections represented the diameter of each glomerulus. The average glomerular diameter of the 10 glomeruli per section was recorded. Additionally, a line connecting the inner and outer edge of Bowman’s capsule parietal epithelium was manually drawn in 12 evenly spaced locations per glomerulus. The average of these 12 lines represented the thickness of Bowman’s capsule parietal epithelium of each glomerulus. The average thickness of the parietal epithelium of the 10 glomeruli per section was recorded.
Statistical Analysis
Quantitative results were expressed as medians with the lower and upper limits (25th and 75th percentiles) of the interquartile range (IQR) for each of the two treatment groups. A Mann–Whitney U-test was performed using KyPlot Software (KyensLab Inc., Tokyo, Japan). Median differences between treatment groups were considered significant at p values =0.05.
Results
Body Weight
Treatment of OVX rats with bFGF for 3 weeks did not significantly affect body weight, a general index of the health of the animals. There was no statistically significant difference between the median body weight of rats before or after treatment with bFGF (342 g [IQR 336 to 352] versus 353 g [IQR 350 to 359], respectively) (p = 0.0693), nor was there a statistically significant difference between the median body weight of vehicle-treated and bFGF-treated rats (357 g [IQR 352 to 360] versus 353 g [IQR 350 to 359], respectively) (p = 0.7129).
Bone Histomorphometry
A detailed analysis of cancellous bone histomorphometry in the lumbar vertebral body from the animals used in the current study has been published previously (Iwaniec et al., 2003). As in the lumbar vertebrae, the proximal tibia of rats treated with bFGF disclosed strong evidence of the bone anabolic effect of bFGF (Table 1). Rats treated with bFGF exhibited a 4-fold increase in median osteoblast surface (p = 0.0029), a 7-fold increase in median osteoid surface (p = 0.0005), and a 67 percent decrease in median osteoclast surface (p = 0.0070) compared with vehicle-treated rats.
Medullary and Extramedullary Hematopoiesis
SC treatment of OVX rats with bFGF caused a 43 percent decrease in the median packed red blood cell volume compared to vehicle-treated rats (p = 0.0002) (Table 2). The reduction in erythrocytes in peripheral circulation was due to a shift toward leukocyte production in the bone marrow, which was reflected by a 61 percent relative increase in the median G:E ratio of bFGF-treated rats compared to vehicle-treated rats (p = 0.0003).
SC treatment of OVX rats with bFGF resulted in a substantial increase in hepatic extramedullary hematopoiesis. Compared to vehicle-treated rats, there was a 29-fold increase in the median number (p ≤ 0.0001) and a 62-fold increase in the median area (p ≤ 0.0001) of hematopoietic foci per hepatic tissue volume. Qualitatively, the hematopoietic foci consisted of almost entirely granulocytic cell, rather than erythroid cell, production (Figure 1E).
Glomerular Morphology and Biochemical Markers of Renal Function
SC treatment of OVX rats with bFGF resulted in mild glomerular hypertrophy (Table 3). There was a significant increase in median glomerular diameter (8% increase; p = ≤ 0.0001) and parietal epithelium thickness (40% increase; p ≤ 0.0001) of bFGF-treated rats compared to vehicle-treated rats. Qualitatively, epithelial cells were more numerous in the glomerular parietal epithelium of bFGF-treated rats (Figure 1 C, D).
There was no compelling serum or urine biochemical evidence of renal dysfunction (Table 3), as indicated by the absence of statistically significant differences between treatment groups in the median serum albumin, creatinine, calcium, and phosphorus concentrations; urine protein-to-creatinine ratio; and urine fractional excretion of phosphorus. However, a markedly elevated urine protein-to-creatinine ratio (12.6, reference value <1.0) was observed in one of the bFGF-treated rats. The median urine fractional excretion of calcium in bFGF-treated rats was decreased by 84% (p = 0.0082) compared to vehicle-treated animals, which was biochemically consistent with the decreased osteoclastic bone resorption observed with bFGF-treatment.
Discussion
To our knowledge, this is the first study to examine select extraskeletal effects of SC bFGF administration in osteopenic OVX rats. The positive bone anabolic effects of bFGF treatment in osteopenic OVX rats were accompanied by moderate to severe anemia and mild glomerular hypertrophy. However, measured biochemical markers of renal function and body weight, a general indicator of health, were not significantly altered by short-term bFGF administration.
The anemia reported here is in accord with that previously documented with systemic (IV and intracardiac) bFGF administration (Mazué, 1992; Lazarous et al., 1995; Iwaniec et al., 2002). Additionally, elevated circulating levels of endogenous bFGF have been correlated with the severity of anemia observed in human patients with solid tumors (Porta et al., 2005). In the present study, histomorphometry was employed to determine a tissue-level mechanism to explain the anemia induced by bFGF administration. Data presented here indicate that bFGF caused a relative increase in granulocytic cell production within the bone marrow which resulted in the observed anemia. Thrombocytopenia, which has been reported in bFGF-treated dogs (Lazarous et al., 1995, 1996; Unger et al., 2000) and previously observed by this research team in bFGF treated-rats (unpublished data), was not investigated in the present study.
When abnormal bone marrow hematopoiesis results in peripheral cytopenias, compensatory extramedullary (non-bone marrow) hematopoiesis may be induced to replace the affected cell lines. In response to decreased bone marrow erythropoiesis and peripheral anemia, as was observed in this study, erythroid extramedullary hematopoiesis would have represented an appropriate physiologic response. However, the opposite was observed in bFGF-treated rats. In addition to increased granulocytic cell production within the bone marrow, bFGF administration induced hepatic extramedullary hematopoiesis that was predominantly granulocytic. It is likely that bFGF administration either indirectly or directly induced granulocytic differentiation of hematopoietic stem cells within the bone marrow and liver or induced proliferation of committed myeloid progenitors at these sites. The latter possibility is suggested by a previous in vitro experiment, which detected FGF receptor mRNA only in lineage-committed myeloid progenitor cells, but not in quiescent, multipotent hematopoietic precursor (stem) cells (Berardi et al., 1995).
The increase in granulocytic cell production reported here is commensurate with previous in vitro studies that employed relatively low doses of bFGF. In long-term human bone marrow cultures, bFGF enhanced granulopoiesis in a dose-dependent manner by modulating the inhibitory response to transforming growth factor-β1 (Wilson et al., 1991; Gabrilove et al., 1993). bFGF increased fibroblast secretion of granulocyte-colony stimulating factor and granulocyte-macrophage colony-stimulating factor in response to interleukin-1 stimulation (Hamilton et al., 1992) and augmented proliferation of granulocyte-macrophage colony-forming units, but not erythroid burst-forming units, in response to granulocyte-macrophage colony-stimulating factor or erythropoietin treatment (Berardi et al., 1995). However, contrary to observations at lower bFGF concentrations, higher doses of bFGF were found to not enhance granulopoiesis in one study (Wilson et al., 1991) and to suppress all hematopoiesis in another study of long-term bone marrow stromal cell cultures (Dooley et al., 1995). Measurement of local cytokine production in the bone marrow of
The mild glomerular hypertrophy observed in this study (increased glomerular diameter and increased thickness of Bowman’s capsule) was consistent with that previously observed in bFGF-treated rats and dogs (Mazué et al., 1992, 1993; Lazarous et al., 1995). In the present study, the mild glomerular hypertrophy detected histologically was not attended by statistically significant alterations in measured biochemical markers of glomerular function. However, in bFGF-treated rats, there was a trend that did not attain statistical significance toward an increased median urine protein-to-creatinine ratio (100 percent increase, p = 0.3711), which is indicative of glomerular protein loss into the urine. This is similar to proteinuria reported previously in bFGF-treated rats and dogs, which was not significantly different between treatment and control groups, but was more severe in those animals that received bFGF, especially at higher doses (Mazué et al., 1992; Unger et al., 2000).
The present study confirmed the bone anabolic effects within the proximal tibia of SC administration of bFGF to osteopenic OVX rats, which were similar to those previously reported in the lumbar vertebra of these animals (Iwaniec et al., 2003). The significantly decreased urine fractional excretion of calcium in bFGF-treated rats reported here was most likely due to decreased bone resorption, as indicated by decreased osteoclast surface in bFGF-treated rats compared to vehicle-treated rats. Renal tubular dysfunction was a much less likely explanation for the observed difference in the urine fractional excretion of calcium, since there was no significant difference between treatment groups in other analytes that are modulated by the renal tubules (i.e., serum calcium, serum phosphorus, or urine fractional excretion of phosphorus).
The strong stimulatory effects of bFGF on bone formation have focused attention on the growth factor as a potential treatment for osteoporosis, but the observed side effects, especially moderate to marked anemia, make its long-term use in humans problematic. However, perhaps the effects of bFGF in tissues other than bone can be minimized by a strategy similar to that applied to prostaglandin E2, which has 4 cell surface receptors (EP 1–4). Yoshida et al. (2002) reported that an EP4-selective agonist has a strong bone anabolic effect in rats without inducing the well-known adverse effects of PGE2 on the gastrointestinal tract. Since bFGF also has 4 cell surface receptors (Ornitz et al., 1996), it is possible that a ligand specific to one of these receptors may induce a bone anabolic response with minimal extraskeletal effects. Such a finding would greatly enhance the potential of bFGF as an osteoporosis therapy.