however, expression and secretion of vegf - a were substantially increased by hypoxia alone or in combination with hyperglycem ia confirming the expected strong response of the cells to their altered environment within the studied time span. angiotensin ii and aldosterone did not modulate vegf - a levels, proving the dominant role of hypoxia in the regulation of the growth factors ' expression and secretion. it was to be expected that hyperglycem ia alone did not modulate vegf - a expression and secretion within 6 h, as possible changes likely manifest only after extended exposure ( 32 ). vegf receptors are expressed in various retinal tissues including the retinal vasculature, mueller cells, and the retinal pigment epithelium ( 33, 34 ). upregulation of vegfr2 in the retinal vasculature is associated with the development of dr and its activation by the ligand vegf - a165 results in elevated permeability of retinal endothelial cells or increased expression of pro - inflammatory mediators in mueller cells in vitro ( 29, 34, 35 ). inhibitors of ace and / or at1, at least in part, prevent vegf - a165 - induced permeability of retinal endothelial cells in vitro and in vivo as well as retinal neovascularization, thereby proving an interaction between both signaling pathways ( 36 - 39 ). however, expression and secretion of vegf - a by mio - m1 cells were not altered by angiotensin ii likely reflecting the different behaviors of both cell types. mrnas coding for proteins of the raas indeed exhibited differential expression patterns in the presence of either angiotensin ii or aldosterone when cells were cultured under hyperglycem ic and / or hypoxic conditions. it is of interest, that angiotensin ii did not alter the expression of its precursor agt under any of the tested conditions, which may indicate that the peptide hormone cannot, directly or indirectly, induce its own expression in mueller cells. to assess a possible pro - inflammatory response of mueller cells, the changes in the expression of mrna as well as secretion of the pro - inflammatory cytokine il - 6, which is constitutively expressed by this cell type ( including mio - m1 cells ), was assessed ( 31 ). however, under normoxic or hypoxic conditions, the amounts of the secreted cytokine and its mrna expression levels were not significantly altered by the treatment with angiotensin ii or aldosterone, suggesting that the hormones do not induce a pro - inflammatory response of the cells under these circumstances. interestingly, aldosterone significantly enhanced the expression of ace mrna under hypoxic conditions, thus not resulting in an inflammatory response, that is enhanced expression or secretion of il - 6 via the angiotensin ii / at1 - axis. angiotensin ii, on the other hand, increased the expression of ace2, resulting in its own inactivation by the formation of angiotensin ( 1 - 7 ), which does not activate at1. similar to the behavior of endothelial cells from the human umbilical cord, the mrna expression levels of il - 6 were increased in mio - m1 cells exposed to hyperglycem ia and aldosterone, although the amount of the secreted cytokine remained unchanged from control cells ( 40 ). however, increased il - 6 expression is likely independent of the angiotensin ii / at1 - axis, as possibly endogenously produced peptide hormone is inactivated by high levels of ace2. although angiotensin ii did not significantly increase il - 6 mrna expression in mio - m1 cells cultured under hyperglycem ic conditions, more il - 6 was secreted under these conditions. higher expression of at1 mrna could lead to stronger activation of the pro - inflammatory angiotensin ii / at1 signaling cascade, similar to that observed for angiotensin ii - activated retinal microglia, which express higher quantities of various pro - inflammatory cytokines and chemok ines including il - 6, a process that is mediated by at1 ( 41 ). elevated permeability of retinal endothelial cells due to il - 6 - mediated trans - signaling in vitro likely contributes to the breakdown of the inner blood - retina barrier in vivo ( 42 ). the protective ace2 / angiotensin ( 1 - 7 ) / mas1 signaling cascade is upregulated during acute and chronic diseases of the heart or kidney to counteract detrimental processes ( 43, 44 ). this signaling cascade seems to also be activated by mueller cells exposed to hyperglycem ia and angiotensin ii, as expression of ace2 and mas1 rna was elevated. thus, the concentrations of the vasodilator angiotensin ( 1 - 7 ) formed by protease ace2 may be higher, and through its interaction with receptor mas1, anti - angiogenic and anti-inflammatory processes can be induced ( 24 - 27 ). however, as mio - m1 cells secreted increased quantities of il - 6 when exposed to hyperglycem ia and angiotensin ii, the pro - inflammatory axis seems to exceed the anti-inflammatory response. a similar inflammatory response to angiotensin ii was also observed after additional exposure of the cells to hyperglycem ia plus hypoxia, as the expression of il - 6 mrna was substantially upregulated. however, the observed lower expression of ace mrna is in line with an assumed capacity of mio - m1 cells to counteract angiotensin ii - induced pro - inflammatory signaling. in vivo, mueller cells, retinal endothelial cells, and retinal pericytes form the so - called neurovascular unit, which tightly regulates vascular homeostasis in the retina ( 45 ). whether the cellular interactions change their individual responses to angiotensin ii could not be evaluated in the present study. however, inhibitors of ace or at1 were found to, at least in part, improve the outcomes of dme in diabetic patients, which supports the findings of the present study that mueller cells likely contribute to angiotensin ii - mediated inflammatory responses present in the early development of this disease ( 46, 47 ). in contrast, the impact of mueller cells on angiotensin ii - mediated inflammatory responses observed in the early development of rvo is likely low when hypoxia plays a dominant role accompanied by induction of expression and secretion of the angiogenic and permeability - inducing growth factor vegf - a ( 9 ). in conclusion, the results of the present in vitro study provide evidence that the responses of mueller cells to activation of the raas by angiotensin ii depend on the environment: a pro - inflammatory response is observed under hyperglycem ic ( plus hypoxic ) conditions, whereas changes induced by hypoxia are not modulated by angiotensin ii. discussion conifer bark, the medicinal use of which dates back more than 2000 years, contains bioactive alkaloids, flavonoids, lignans, phenolic acids, proanthocyanidins, stilbenes, and terpenoids that contribute to its therapeutic potential. various conifer bark extracts are used nowadays in the nutraceutical, food, and pharmaceutical industries because of their health - promoting effects in numerous ailments and diseases [ 27, 28, 29 ]. such extracts are pycnogenol®, flavangenol®, and oligopin® derived from pinus pinaster ait. bark [ 29 ], enzogenol® produced from pinus radiata d. don bark [ 30 ], and abigenol® originating from abies alba mill. bark [ 31 ]. pinus cembra l. ( pinaceae, swiss stone pine, arolla pine, cembran pine, cedar pine ) is a coniferous tree growing in the alps and carpathian mountains [ 32 ]. the bark has been scarcely investigated for its chemical composition and biological activity. we have previously assessed the antioxidant potential of the raw bark extract ( 80 % methanolic bark extract ) and found ec50 values of 71. 1 ± 0. 5 and 26. 0 ± 0. 3 μg / ml in the dpph radical scavenging and reducing power assays, respectively. the antioxidant potential of the raw bark extract is strongly associated with the total phenolic content, quantified as 299. 3 ± 1. 4 mg / g. in the same assays, catechin was more effective ( ec50 = 5. 56 ± 0. 05 and 3. 70 ± 0. 03 μg / ml, respectively ) [ 26 ].