Downstream exposure to growth factors causes elevated velocity and dilation in arteriolar networks

Abstract

Our goal was to characterize changes in flow and diameter with vascular endothelial cell growth factor A (VEGF-A) and fibroblast growth factor 2 (FGF2). Observations were made in arteriolar networks of the cheek pouch tissue in anesthetized hamsters (pentobarbital 70 mg/kg, i.p., n = 45). Local and remote dilation responses to micropipette-applied VEGF or FGF2 yielded similar EC(50) values. The role of gap junctions in the remote response was tested by applying sucrose, halothane or 18αGA to the feed arteriole midway between the remote stimulation and upstream observation sites; all remote dilation to FGF2 was prevented, while only the early dilation to VEGF was blocked. The remote dilation to VEGF displayed a second rheologic mechanism. The second mechanism involved an abrupt increase in upstream velocity and shear rate, followed by nitro-arginine sensitive dilation. To test whether the abrupt increase in shear could be caused by other agents known to cause edema, remote responses to histamine and thrombin were tested. Each caused an abrupt increase in velocity followed by nitro-arginine-sensitive dilation. This study shows that VEGF or agents that increase permeability can initiate an upstream velocity increase with dilation that recruits flow to the network; this is in addition to simultaneous gap junction-mediated dilation.

Fig. 1

Shown is a schematic of the arteriolar circulation in the hamster cheek pouch, with an expanded view of the arteriolar network. Multiple arteriolar networks arise from the extensive arcading system. All observations of diameter and blood flow were made at location A. All test agents were applied via micropipette. Test agents were applied to location A, the entrance to the network to obtain local responses (protocol 1, Methods). Test agents were applied to location C to obtain remote responses (protocol 2). To test the involvement of gap junctional communication in these responses, the gap junction inhibitors were applied to location B (protocol 3). To test the involvement of flow-mediated dilation at location A, LNNA (nitro-arginine) was applied to location A (protocol 4).

Fig. 2

Shown is the normalized change in diameter [1 + (peak – baseline)/baseline] for the local (a) and remote (b) responses to growth factors. The x-axis shows the concentration in the micropipette. a Local response relationships for VEGF (n = 9) and for FGF2 (n = 7), using protocol 1. b Remote dilation response relationships for VEGF (n = 9) and FGF2 (n = 10), using protocol 2. The fitted EC₅₀ and maximal values are in table 2. Data are presented as means ± SEM.

Fig. 3

Shown are the normalized remote velocity, diameter and shear rate values [1 + (peak – baseline)/baseline] as a function of time with downstream VEGF (a, n = 11) or FGF2 (b, n = 7) at 1,000 ng/ml in the micropipette. Data was binned for each 5 s; error bars intentionally left off to observe trends. A 30-second baseline preceded a 60-second remote exposure; exposure onset is at 0 s on this graph.

Fig. 4

Shown is the normalized remote shear rate [1 + (peak – baseline)/baseline] upstream with downstream application of growth factors, using protocol 2. The x-axis shows the concentration in the micropipette. Early (a) and late (b) responses for VEGF (n = 6) and for FGF2 (n = 6), as defined in figure 3a. The EC₅₀ and maximal values are in table 2. * Significantly less than baseline. a 1,000 ng/ml FGF2 does not differ from baseline (p = 0.09). Data are presented as means ± SEM.

Fig. 5

Shown is the normalized change in red blood cell flux [1 + (peak – baseline)/baseline] into the network with downstream application of VEGF (n = 6) or FGF2 (n = 6), using protocol 2. The x-axis shows the concentration in the micropipette. Data are presented as means ± SEM.

Fig. 6

Shown is the normalized change in diameter [1 + (peak – baseline)/baseline] for the remote dilation to growth factors with and without gap junction inhibition, using protocol 3. Early and late dilation responses are noted for 100 ng/ml VEGF (a, n = 7) or FGF2 (b, n = 6) during control, or simultaneously with a gap junction inhibitor: sucrose, 150 mOsm sucrose plus control suffusate; halothane, 3.4 mM; 18αGA, 50 μM. * Differs from early control; ⁺ differs from late control. Data are presented as means ± SEM.

Fig. 7

Shown is the normalized change in diameter [1 + (peak – baseline)/baseline] for the remote dilation response to growth factors with and without LNNA, using protocol 4. Early and late dilation responses are noted for 100 ng/ml VEGF (a, n = 6) or FGF2 (b, n = 5) during control, or simultaneously with LNNA (50 μM). * Differs from early. Data are presented as means ± SEM.

Fig. 8

Shown are the normalized velocity, diameter and shear rate values [1 + (peak – baseline)/baseline] as a function of time with histamine (a, 50 μM, n = 10) and thrombin (b, 5 U/ml, n = 10). Data was binned for each 5 s; error bars intentionally left off to observe trends. The values are normalized to the average baseline. A 30-second baseline preceded a 60-second remote exposure (at 30 s on this graph).