BACKGROUND
insulin-like growth factors are peptide growth factors and have been found to be powerful mitogens for intestinal epithelial cells with modulation by six specific igf binding proteins   <cit> .
the igfbp species have been shown to inhibit or potentiate the proliferative response to igfs, depending on the cell type and culture conditions  <cit> .
the primary igf found in serum during prenatal development is igf-ii  <cit> .
igf-ii mrna levels in the human duodenum were also found to be five times higher in the fetus than in the child  <cit> .
the igfbp- <dig> which is normally a negative modulator of growth also has a developmental pattern of expression.
a study that examined  <dig> normal fibroblast cell lines created from subjects ranging from fetal to  <dig> years of age found progressively increasing amounts of igfbp- <dig> production with age  <cit> .
this direct correlation of donor age and igfbp- <dig> levels was consistent during all phases of fibroblast cell growth  <cit> .
fetal sheep have high circulating igfbp- <dig> serum levels that peak at approximately 70%  of gestation, then begin to fall  <cit> .
in studies measuring tissue specific mrna levels, fetal tissue igfbp- <dig> follows a pattern similar to serum in fetal sheep  <cit> .
another study, comparing post-natal intestinal and adult total intestinal mrna for the igfbps, found higher mrna post-natal levels of igfbps- <dig> through  <dig> that fell with age.
<cit> .
the fibroblasts igf axis is important in the intestine because of the paracrine effect on the intestinal mucosa.
a dual culture model found that intestinal epithelial cells grown on intestinal fibroblasts had greater proliferation when stimulated by the paracrine effect of fibroblast produced igf-ii  <cit> .
a transgenic mouse model with igf-i overexpression coupled with a mesenchymal promoter was found to have increased igf-i overexpression in the gastrointestinal lamina propria this resulted in a mucosa with increased dna and protein content manifested by increased crypt cell mitosis and sucrase activity  <cit> .
this study was designed to compare the regulation of the igf-axis and the igf responses of fetal and adult intestinal fibroblasts.
the hypothesis was that fetal small intestinal fibroblasts would have a higher production and respond better to igf-ii than adult intestinal fibroblasts.
we also expected to find the igfbps levels present to decrease to allow increased proliferation in the fetal intestinal fibroblasts.
RESULTS
protein assay of conditioned medium sample
the media conditioned by the cultured maternal or fetal intestinal fibroblasts was collected at confluence and post-confluence.
the conditioned media was stored at -20°c until analysis.
the measured protein concentrations did not vary significantly for the maternal or fetal fibroblasts comparing confluence or post-confluence.
there was also no significant difference between maternal or fetal conditioned media at either time point measured .
igf-i and igf-ii expression
the conditioned media from the maternal and fetal intestinal fibroblasts was assayed for igf concentration by radioimmunoassays.
the maternal and fetal intestinal fibroblasts produced primarily igf-ii.
at confluence, the fetal fibroblasts produced greater concentrations of igf-i and igf-ii than maternal fibroblasts, but this did not achieve statistical significance  .
one week post-confluent fetal fibroblasts had increased igf-i secretion, while that of the maternal fibroblasts decreased .
the igf-ii secretion was decreased slightly one week post-confluence in fetal fibroblasts, and it was significantly decreased in maternal fibroblasts .
ligand blotting for igfbp expression
ligand blotting utilizes the high affinity of the igfbps for the igfs to detect the presence of igfbp species present in the conditioned media.
three igfbp bands were seen consistently with relative molecular weights that have been characterized for ovine igfbps- <dig> , - <dig>  and - <dig>   <cit> .
immunoblotting was performed and verified that the  <dig> kda and  <dig> kda bands were igfbp- <dig> and - <dig> respectively .
the fibroblasts produced primarily igfbp- <dig> in both maternal and fetal cell lines.
at confluence, there were no significant differences in the expression of the igfbps between the fetal and maternal fibroblasts .
fetal fibroblasts had non-significant changes in expression of igfbp- <dig> one-week post-confluence  and nearly significant reductions in igfbp- <dig> .
in contrast, maternal fibroblasts post-confluence had nearly significant increased igfbp- <dig>  and significantly increased igfbp- <dig> .
there were no significant changes in any of the igfbp- <dig> levels measured.
the increases in igfbp- <dig> and igfbp- <dig> in the post-confluent maternal fibroblasts were significant when compared to the post-confluent fetal fibroblasts .
fibroblast response to igf-i and igf-ii
maternal and fetal fibroblasts were grown to confluence and then treated with  <dig> nm recombinant human igf-i or igf-ii.
the mtt assay demonstrated that all populations of fibroblasts responded to igf treatment with statistically significant  greater cell numbers.
a percentage was calculated for graphic presentation, for statistical calculations the actual measured absorbance at  <dig> nm was utilized.
triplicate wells of each experimental condition was undertaken for each of the cell lines.
maternal fibroblasts responded with greater cell numbers for igf-i than igf-ii compared to controls .
fetal fibroblasts responded to igf-ii better than igf-i, although not significantly, and at the same rate that maternal fibroblasts responded to igf-i .
the amount of increase in the fetal cells in response to igf-ii was significantly greater that the amount of increase seen in the maternal cells .
discussion
the primary finding from our studies is the increased responsiveness of the fetal intestinal fibroblasts to igf-ii compared to the adult intestinal fibroblasts.
the adult intestinal fibroblasts responded to igf-ii with only a 55% increase in the cell numbers compared to controls.
the fetal intestinal fibroblasts had the maximum response to igf-ii with an 88% increase in cell numbers.
interestingly, this is the same rate at which the adult intestinal fibroblasts responded to igf-i.
circulating igf-i levels are low in fetal sheep and increase throughout gestation, while circulating igf-ii and small intestine igf-ii mrna levels are much higher and increase to a peak late in gestation  <cit> .
the primary prenatal igf in mice and humans is igf-ii as well, but in adults the primary circulating igf is igf-i  <cit> .
therefore, our study suggests that the intestinal fibroblast proliferation response to the igfs is developmentally regulated for the maximum response dependent on developmental stage of the individual.
comparing the igf concentrations used to stimulate fibroblast proliferation to physiologic conditions, bloomfield et al.
reported igf-i concentrations in sheep amniotic fluid of  <dig>  nm at 110– <dig> days gestation  <cit> .
a paper from blahovec et al.
reported that the amniotic fluid at  <dig> days gestation had concentrations that were roughly <dig>  nm igf-i and  <dig>  nm igf-ii; subsequent western blotting for igf-ii throughout gestation showed increasing concentrations until it peaked between  <dig> and  <dig> days gestation  <cit> .
studies have shown poor absorption of enteral igfs after birth but there are several studies in fetal animals indicating significant absorption of ingested igfs  <cit> .
the  <dig> nm concentration utilized in these experiments correlates well with the reported igf-i and -ii concentrations in porcine milk  during the immediate post-partum period  <cit> .
the fetal intestinal fibroblast would be expected to be stimulated by approximately this concentration of igfs from both serum and luminal sources.
both maternal and fetal intestinal fibroblasts produced primarily igf-ii.
the fetal intestinal fibroblasts maintained a relatively high level of igf expression even when post-confluent while the maternal intestinal fibroblasts had significant decreases in igf levels.
this is consistent with human studies which found greater levels of igf-ii mrna in the duodenum in fetal intestine compared with children and adults  <cit> .
this study also found the total intestinal mrna for igf-i was higher than for igf-ii in the adult  <cit> .
a study characterizing the igf production in a human colon fibroblast cell line and primary culture adult small intestinal fibroblasts found significant igf-ii and low levels of igf-i mrna  <cit> .
our cell culture data that intestinal fibroblasts produce primarily igf-ii and that the greatest level of production is by the fetal intestinal fibroblasts is consistent with the literature mrna studies.
the igfbp findings were not what we hypothesized.
the fetal intestinal fibroblasts had no significant changes when post-confluent.
this is in comparison to the highly significant increase in igfbp- <dig> seen in the post-confluent maternal intestinal fibroblasts.
fetal rat lung fibroblasts plated near confluence had increased levels of igfbps - <dig> and - <dig> and stable igfbp- <dig> levels after  <dig> hours of culture in serum-free media, similar to our conditions  <cit> .
an older study that examined igfbp production by developmental stage; found that fetal sheep skin fibroblasts had relatively high levels of igfbp production  <cit> .
the igfbps produced by the fetal skin fibroblasts were reported by relative molecular weight to be consistent with igfbps - <dig> and - <dig> compared to low levels of igfbp- <dig>  <cit> .
the concentration of the igfbp with a relative molecular weight consistent with igfbp- <dig> had a marked increase in the perinatal period  <cit> .
a study by goldstein et al that examined igfbp- <dig> in  <dig> normal fibroblast cell lines  found increasing igfbp- <dig> levels that correlated with the donors chronological age  <cit> .
this study found increased igfbp- <dig> levels at all age levels with increased culture time  <cit> .
the significant increase in production of igfbp- <dig> by the maternal intestinal fibroblasts is very consistent with the pattern seen in these other fibroblast cell lines  <cit> .
the lack of a significant change in igfbp levels in the fetal intestinal fibroblasts is in contrast to the increased levels seen in the other studies of fetal fibroblasts  <cit> .
this lack of increase could be speculated as significant finding because it would allow continued stimulation of the fetal intestinal fibroblasts by the igfs present.
the fetal intestinal fibroblasts are important in the development of the intestinal mucosa.
a transgenic mouse model with a mesenchymal promoter linked to an igf-i gene found increased igf-i overexpression in the gastrointestinal lamina propria resulted in increased crypt cell mitosis and sucrase activity  <cit> .
this evidence would suggest a role for paracrine stimulation by the fetal intestinal fibroblast produced igfs on the intestinal mucosa.
the persistent production of igfs by the fetal intestinal fibroblasts suggests the developmental regulation of igf expression for paracrine stimulation of the fetal intestinal mucosa.
CONCLUSIONS
in this study, we always found a higher production of both igfs by fetal intestinal fibroblasts compared to maternal intestinal fibroblasts.
we also demonstrated increased proliferation by fetal compared to adult intestinal fibroblasts in response to igf-ii, the primary circulating igf in the fetus.

 insulin-like growth factors are peptide growth factors and have been found to be powerful mitogens for intestinal epithelial cells with modulation by six specific igf binding proteins. the igfbp species have been shown to inhibit or potentiate the proliferative response to igfs, depending on the cell type and culture conditions. the primary igf found in serum during prenatal development is igf-ii. igf-ii mrna levels in the human duodenum were also found to be five times higher in the fetus than in the child. the igfbp- which is normally a negative modulator of growth also has a developmental pattern of expression. a study that examined normal fibroblast cell lines created from subjects ranging from fetal to years of age found progressively increasing amounts of igfbp- production with age. this direct correlation of donor age and igfbp- levels was consistent during all phases of fibroblast cell growth. fetal sheep have high circulating igfbp- serum levels that peak at approximately 70% of gestation, then begin to fall. in studies measuring tissue specific mrna levels, fetal tissue igfbp- follows a pattern similar to serum in fetal sheep. another study, comparing post-natal intestinal and adult total intestinal mrna for the igfbps, found higher mrna post-natal levels of igfbps- through that fell with age.. the fibroblasts igf axis is important in the intestine because of the paracrine effect on the intestinal mucosa. a dual culture model found that intestinal epithelial cells grown on intestinal fibroblasts had greater proliferation when stimulated by the paracrine effect of fibroblast produced igf-ii. a transgenic mouse model with igf-i overexpression coupled with a mesenchymal promoter was found to have increased igf-i overexpression in the gastrointestinal lamina propria this resulted in a mucosa with increased dna and protein content manifested by increased crypt cell mitosis and sucrase activity. this study was designed to compare the regulation of the igf-axis and the igf responses of fetal and adult intestinal fibroblasts. the hypothesis was that fetal small intestinal fibroblasts would have a higher production and respond better to igf-ii than adult intestinal fibroblasts. we also expected to find the igfbps levels present to decrease to allow increased proliferation in the fetal intestinal fibroblasts.  protein assay of conditioned medium sample the media conditioned by the cultured maternal or fetal intestinal fibroblasts was collected at confluence and post-confluence. the conditioned media was stored at -20°c until analysis. the measured protein concentrations did not vary significantly for the maternal or fetal fibroblasts comparing confluence or post-confluence. there was also no significant difference between maternal or fetal conditioned media at either time point measured. igf-i and igf-ii expression the conditioned media from the maternal and fetal intestinal fibroblasts was assayed for igf concentration by radioimmunoassays. the maternal and fetal intestinal fibroblasts produced primarily igf-ii. at confluence, the fetal fibroblasts produced greater concentrations of igf-i and igf-ii than maternal fibroblasts, but this did not achieve statistical significance. one week post-confluent fetal fibroblasts had increased igf-i secretion, while that of the maternal fibroblasts decreased. the igf-ii secretion was decreased slightly one week post-confluence in fetal fibroblasts, and it was significantly decreased in maternal fibroblasts. ligand blotting for igfbp expression ligand blotting utilizes the high affinity of the igfbps for the igfs to detect the presence of igfbp species present in the conditioned media. three igfbp bands were seen consistently with relative molecular weights that have been characterized for ovine igfbps-, - and -. immunoblotting was performed and verified that the kda and kda bands were igfbp- and - respectively. the fibroblasts produced primarily igfbp- in both maternal and fetal cell lines. at confluence, there were no significant differences in the expression of the igfbps between the fetal and maternal fibroblasts. fetal fibroblasts had non-significant changes in expression of igfbp- one-week post-confluence and nearly significant reductions in igfbp-. in contrast, maternal fibroblasts post-confluence had nearly significant increased igfbp- and significantly increased igfbp-. there were no significant changes in any of the igfbp- levels measured. the increases in igfbp- and igfbp- in the post-confluent maternal fibroblasts were significant when compared to the post-confluent fetal fibroblasts. fibroblast response to igf-i and igf-ii maternal and fetal fibroblasts were grown to confluence and then treated with nm recombinant human igf-i or igf-ii. the mtt assay demonstrated that all populations of fibroblasts responded to igf treatment with statistically significant greater cell numbers. a percentage was calculated for graphic presentation, for statistical calculations the actual measured absorbance at nm was utilized. triplicate wells of each experimental condition was undertaken for each of the cell lines. maternal fibroblasts responded with greater cell numbers for igf-i than igf-ii compared to controls. fetal fibroblasts responded to igf-ii better than igf-i, although not significantly, and at the same rate that maternal fibroblasts responded to igf-i. the amount of increase in the fetal cells in response to igf-ii was significantly greater that the amount of increase seen in the maternal cells. discussion the primary finding from our studies is the increased responsiveness of the fetal intestinal fibroblasts to igf-ii compared to the adult intestinal fibroblasts. the adult intestinal fibroblasts responded to igf-ii with only a 55% increase in the cell numbers compared to controls. the fetal intestinal fibroblasts had the maximum response to igf-ii with an 88% increase in cell numbers. interestingly, this is the same rate at which the adult intestinal fibroblasts responded to igf-i. circulating igf-i levels are low in fetal sheep and increase throughout gestation, while circulating igf-ii and small intestine igf-ii mrna levels are much higher and increase to a peak late in gestation. the primary prenatal igf in mice and humans is igf-ii as well, but in adults the primary circulating igf is igf-i. therefore, our study suggests that the intestinal fibroblast proliferation response to the igfs is developmentally regulated for the maximum response dependent on developmental stage of the individual. comparing the igf concentrations used to stimulate fibroblast proliferation to physiologic conditions, bloomfield et al. reported igf-i concentrations in sheep amniotic fluid of nm at 110– days gestation. a paper from blahovec et al. reported that the amniotic fluid at days gestation had concentrations that were roughly nm igf-i and nm igf-ii; subsequent western blotting for igf-ii throughout gestation showed increasing concentrations until it peaked between and days gestation. studies have shown poor absorption of enteral igfs after birth but there are several studies in fetal animals indicating significant absorption of ingested igfs. the nm concentration utilized in these experiments correlates well with the reported igf-i and -ii concentrations in porcine milk during the immediate post-partum period. the fetal intestinal fibroblast would be expected to be stimulated by approximately this concentration of igfs from both serum and luminal sources. both maternal and fetal intestinal fibroblasts produced primarily igf-ii. the fetal intestinal fibroblasts maintained a relatively high level of igf expression even when post-confluent while the maternal intestinal fibroblasts had significant decreases in igf levels. this is consistent with human studies which found greater levels of igf-ii mrna in the duodenum in fetal intestine compared with children and adults. this study also found the total intestinal mrna for igf-i was higher than for igf-ii in the adult. a study characterizing the igf production in a human colon fibroblast cell line and primary culture adult small intestinal fibroblasts found significant igf-ii and low levels of igf-i mrna. our cell culture data that intestinal fibroblasts produce primarily igf-ii and that the greatest level of production is by the fetal intestinal fibroblasts is consistent with the literature mrna studies. the igfbp findings were not what we hypothesized. the fetal intestinal fibroblasts had no significant changes when post-confluent. this is in comparison to the highly significant increase in igfbp- seen in the post-confluent maternal intestinal fibroblasts. fetal rat lung fibroblasts plated near confluence had increased levels of igfbps - and - and stable igfbp- levels after hours of culture in serum-free media, similar to our conditions. an older study that examined igfbp production by developmental stage; found that fetal sheep skin fibroblasts had relatively high levels of igfbp production. the igfbps produced by the fetal skin fibroblasts were reported by relative molecular weight to be consistent with igfbps - and - compared to low levels of igfbp-. the concentration of the igfbp with a relative molecular weight consistent with igfbp- had a marked increase in the perinatal period. a study by goldstein et al that examined igfbp- in normal fibroblast cell lines found increasing igfbp- levels that correlated with the donors chronological age. this study found increased igfbp- levels at all age levels with increased culture time. the significant increase in production of igfbp- by the maternal intestinal fibroblasts is very consistent with the pattern seen in these other fibroblast cell lines. the lack of a significant change in igfbp levels in the fetal intestinal fibroblasts is in contrast to the increased levels seen in the other studies of fetal fibroblasts. this lack of increase could be speculated as significant finding because it would allow continued stimulation of the fetal intestinal fibroblasts by the igfs present. the fetal intestinal fibroblasts are important in the development of the intestinal mucosa. a transgenic mouse model with a mesenchymal promoter linked to an igf-i gene found increased igf-i overexpression in the gastrointestinal lamina propria resulted in increased crypt cell mitosis and sucrase activity. this evidence would suggest a role for paracrine stimulation by the fetal intestinal fibroblast produced igfs on the intestinal mucosa. the persistent production of igfs by the fetal intestinal fibroblasts suggests the developmental regulation of igf expression for paracrine stimulation of the fetal intestinal mucosa.  in this study, we always found a higher production of both igfs by fetal intestinal fibroblasts compared to maternal intestinal fibroblasts. we also demonstrated increased proliferation by fetal compared to adult intestinal fibroblasts in response to igf-ii, the primary circulating igf in the fetus. 
post-confluence, fetal fibroblasts had no significant changes in igfbp- and igfbp- expression. post-confluent maternal fibroblasts had increased igfbp- levels that were significant compared to the fetal fibroblasts. igf-i increased in post-confluent fetal fibroblasts, while in maternal fibroblasts it decreased. fetal fibroblasts responded to igf-ii slightly better than igf-i and significantly greater than maternal cells. we compared igf responses of fetal and adult intestinal fibroblasts to identify a developmental difference in the igf-axis. intestinal fibroblasts were isolated from maternal and fetal jejunum. fetal intestinal fibroblasts respond to igf-ii with greater proliferation and do not have the increased igfbps seen post-confluence in adult intestinal fibroblasts. igf-ii secretion decreased significantly in post-confluent maternal fibroblasts. there were no significant differences in igfbp expression at confluence. the proliferative response at confluence to nm igf-i or -ii was compared. maternal fibroblasts proliferated more with igf-i than igf-ii.
 we compared igf responses of fetal and adult intestinal fibroblasts to identify a developmental difference in the igf-axis. intestinal fibroblasts were isolated from maternal and fetal jejunum. media was conditioned at confluence and one week afterwards. the proliferative response at confluence to nm igf-i or -ii was compared.  there were no significant differences in igfbp expression at confluence. post-confluence, fetal fibroblasts had no significant changes in igfbp- and igfbp- expression. post-confluent maternal fibroblasts had increased igfbp- levels that were significant compared to the fetal fibroblasts. igf-i increased in post-confluent fetal fibroblasts, while in maternal fibroblasts it decreased. igf-ii secretion decreased significantly in post-confluent maternal fibroblasts. maternal fibroblasts proliferated more with igf-i than igf-ii. fetal fibroblasts responded to igf-ii slightly better than igf-i and significantly greater than maternal cells.  fetal intestinal fibroblasts respond to igf-ii with greater proliferation and do not have the increased igfbps seen post-confluence in adult intestinal fibroblasts. 