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J Appl Physiol 102: 2279-2287, 2007. First published March 22, 2007; doi:10.1152/japplphysiol.00537.2006
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Mechanisms initiating integrin-stimulated flow recruitment in arteriolar networks

Mary D. Frame,1 Richard J. Rivers,2 Owen Altland,3 and Scott Cameron3

1Stony Brook University, Stony Brook, New York; 2Johns Hopkins University, Baltimore, Maryland; and 3University of Rochester, Rochester, New York

Submitted 12 May 2006 ; accepted in final form 19 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Our purpose was to investigate the local mechanisms involved in network-wide flow and diameter changes observed with localized downstream vitronectin receptor ligation; we tested specific K or Cl channels known to be involved in either dilation or elevated permeability following vitronectin receptor activation and tested integrin-linked pathway elements of tyrosine phosphorylation and protein kinase C (PKC). Arteriolar networks were observed in the cheek pouch tissue of anesthetized (pentobarbital sodium, 70 mg/kg) hamsters (n = 86) using intravital microscopy. Terminal arteriolar branches of the networks were stimulated with micropipette LM609 (0.5–10 µg/ml, 60 s) alone or with inhibitors (separate micropipette). Hemodynamic changes (diameter, red blood cell flux, velocity) were observed at the upstream entrance to the network. LM609 alone stimulated first an increase in wall shear stress (WSS), followed by a dilation that recovered WSS to baseline or below. K channel inhibition (glybenclamide, 4-AP) had no effect on the initial peak in WSS, but decreased remote vasodilation. Cl channel inhibition (DIDS, IAA-94, niflumic acid) or inhibition of PKC (chelerythrine) prevented the initial peak in WSS and decreased remote vasodilation. Inhibition of tyrosine phosphorylation (genistein) prevented both. With the use of nitro-arginine at the observation site, the initial peak in WSS was not affected, but remote vasodilation was decreased. We conclude the remote response consists of an initial peak in WSS that relies on both PKC activity and depolarization downstream, leading to an upstream flow mediated dilation and a secondary remote dilation that relies on hyperpolarization downstream at the stimulus site; both components require tyrosine phosphorylation downstream.

flow-dependent dilation; remote responses; vascular communication

PERIPHERAL FLOW RECRUITMENT has been thought to be largely controlled by the caliber of terminal arterioles, at rest (20, 29, 30, 46), during reperfusion following ischemia (29), or during induced hyperemia (40, 41, 47), as well as in many metabolic and autonomic states (1, 3, 13, 34). A question that we address is how do the responses of individual arteriolar segments become coordinated and at what point do the responses of individual arteriolar segments reach the transition point, the point at which the responses of the individual vessel segments are coordinated to now describe organ level flow? One way that the segment responses could transition to the larger and far-reaching organ level responses could be via vascular communication of remote responses in a manner that would predictably alter flow and shear patterns within anatomically distinct groups of vessels (5, 15, 18, 39, 42, 44).

Our prior study (17) outlines an integrative mechanism for microvascular flow recruitment, which is stimulated by an interaction between the vessel wall and pharmacologic factors mimicking dynamic interaction with the surrounding matrix. Specifically, localized downstream stimulation of the {alpha}vbeta3 integrin (vitronectin) receptor, abluminally, induces an abrupt remote (~1,000 µm upstream) elevation of flow, followed by a remote dilation; the remote dilation is at least partially flow mediated and linked to vascular endothelial cell growth factor receptor 2 tyrosine phosphorylation (19, 26). The total response recruits flow quite specifically to only the downstream branch arteriole that is stimulated. The important observations here are that remote flow increases into an entire arteriolar network temporally before any changes in upstream resistance (diameter), and the flow that is recruited is specifically directed to the stimulated location of the network bypassing other flow paths. This means that the upstream portions of the network are responding to a programmed flow recruitment event requiring integration of signals from the arteriolar network as a whole.

The present study focuses on the initiation events that stimulate this integrative hemodynamic response. In a prior study, we determined that ligation of the vitronectin receptor with the {alpha}vbeta3 integrin antibody LM609 on terminal arterioles (not capillary endothelium) was essential for the response (17, 19). The integrin-associated signal transduction cascade in vascular cells appears to exhibit many convergent mechanisms for shear/flow linked, angiogenic (i.e., matrix interaction), and permeability inducing pathways. Key phosphorylation points include tyrosine phosphorylation of p125FAK (focal adhesion kinase) and, separately, a nitric oxide-linked pathway (43). Furthermore, many of the known integrin-stimulated pathways in vascular cells include an essential role for protein kinase C (PKC) activation in mediating permeability changes (2, 50). In isolated vessels, cellular mediators of local arteriolar dilation through this pathway are thought to require chloride channel activation, with a modulating role for ATP-sensitive potassium channels to regulate calcium metabolism (11, 31). The objective of this study was to investigate the signal transduction pathway that initiates the remote flow recruitment response to vitronectin receptor ligation. We hypothesized that tyrosine phosphorylation and PKC activity would be required to initiate the total response downstream. Furthermore, we expected that specific chloride or potassium channels that are associated with vitronectin receptor ligation would also be required to mimic the physiological response.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Experimental animal care and procedures.   With University approval (UCAR, University of Rochester; IACUC, Stony Brook University), adult male Golden hamsters [HSD:Syr, 82 ± 11 days, 116 ± 12 g (mean ± SD), n = 86] were anesthetized with pentobarbital sodium (hamsters: 70 mg/kg ip). Body temperature was maintained between 37° and 38°C. The hamsters were tracheostomized, and a right jugular catheter was placed for administration of the fluorescently labeled red blood cells. The left cheek pouch was prepared for in situ microcirculatory observations. The preparation was continuously superfused with bicarbonate buffered saline containing (in mM) 132 NaCl, 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, 20 NaHCO3 (equilibrated with gas containing 5% CO2 and 95% N2 gas, pH 7.4 at 34°C). All chemicals were obtained from Sigma Chemical (St. Louis, MO), unless otherwise noted. Fluorescently labeled red blood cells (substituted tetramethyl rhodamine isothiocyanate, XRITC, Molecular Probes, Eugene, OR), were from age and weight-matched donor animals (n = 41), as previously described (17, 20).

During a 60-min stabilization period, fluorescently labeled red blood cells were administered and arteriolar tone was verified by dilation to topically applied 10–4 M adenosine, and constriction to 10% oxygen gas added to the superfusate. Observations were made along an arteriolar network consisting of a feed with three- to six-branch arterioles, located as described previously (17, 20). The microcirculation was observed with trans-illumination using a modified Nikon upright microscope (Nikon, Japan), with a x25 (Nikon) or a x40 (SWI, Olympus) objective. Video images were produced using a CCD 72s video camera and a Gen/Sys II video intensifier (Dage-MTI, Michigan City, IN) or an ICCD Mega-10 intensified video camera (Solamere Technology Group, Salt Lake City, UT). Responses were videorecorded using a Panasonic AG-7350 SVHS videotape.

Micropipette stimulation protocol.   The downstream branch arteriole of an arteriolar network was exposed to micropipette delivered test agents. The micropipette tip diameters were 16 ± 3 µm. Micropipettes were placed within 25 µm of the vessel wall. All agents were applied using a pressure delivery system with –0.2 psi holding pressure and +0.3 psi delivery pressure. Each micropipette contained 25–50 µM fluorescein-labeled dextran (4,000 mol wt) to verify regions that were exposed (observed using a Chroma B1E filter, Chroma).

Data collection protocol.   Remote responses to LM609 (anti-{alpha}vbeta3 monoclonal antibody, Chemicon International) were tested based on our published finding that this agent elicits an increase in remote velocity prior to remote dilation (19). Baseline diameter red blood cell flux and velocity were obtained for the 30 s immediately prior to downstream exposure and for the 60-s stimulation time period. The control remote responses were initiated by 60-s exposure to LM609, methacholine, or pinacidil (KATP agonist, ATP-sensitive potassium channel; concentrations noted on the figures or in the legends). Diameters were analyzed both online and offline from the recorded image; red blood cell velocity and flux were analyzed offline. The baseline velocity met criteria of steady state, as previously described (19). [The LM609 response is robust, and with 5-min recovery between responses, up to 16 exposures have produced identical responses at one arteriolar network (19). For the present study, a maximum of six exposures per network were performed. In some protocols, two networks were studied per animal.]

After these control responses, inhibitor agents were applied for 5–30 min prior to LM609, methacholine, or pinacidil application and continuously during the response. The inhibitor concentrations varied by protocol and are given in the text and figure legends. Only one inhibitor agent was tested per animal with randomized concentrations. A range of K and Cl channel antagonists were chosen based on the proposed action of integrins on local dilation [linked to KATP or ClCa channels, (35)] or on local changes in membrane potential [linked to voltage or Ca-dependent K or Cl channels (12, 27)]. Calcium-dependent chloride channels are more specifically blocked with niflumic acid (µM range), whereas voltage-dependent chloride channels are blocked by DIDS (µM range) or indanyloxyacetic acid 94 (IAA-94, µM range) vs. the less-specific niflumic acid (mM range; 22, 23). Additionally, the ATP-sensitive chloride channels are blocked by niflumic acid or by glybenclamide (mM range for both). Glybenclamide (µM range), and, to a lesser degree, niflumic acid, blocks ATP-sensitive potassium channels, whereas 4-aminopyridine (4-AP, µM range) blocks the voltage-dependent potassium channels.

Inhibitor agents were tested as follows: glybenclamide (in DMSO), 4-AP, DIDS, IAA-94, or niflumic acid was applied for 5 min prior to and during LM609, methacholine, or pinacidil exposure. The role of protein kinase C was tested with chelerythrine (likewise applied for 5 min); efficacy was tested with 0.1 µg/ml phorbol 12-myristate 13-acetate, PMA. Tyrosine kinase activity was tested with genistein (in ethanol) and the inactive analog, diadzein (in ethanol); genistein or diadzein were applied for 30 min prior to and during LM609 exposure. One inhibitor agent, N{omega}-nitro-L-arginine (L-NNA) was micropipette applied to the upstream observation site, with remote responses to LM609 determined 20 min later. L-NNA was used upstream to test whether a component of the LM609 response required endogenous NO and could thus be linked to a flow-mediated mechanism.

Data collection and analysis.   The primary data endpoints of diameter, red blood cell flux and velocity were collected from the recorded image. Additionally, all diameters were measures online using a video caliper system (Microvascular Research Institute, College Station, TX) and a software data acquisition system (Strawberry Tree, Workbench, Sunnyvale, CA), calibrated with a micrometer. For each exposure, data were pooled for the 30-s baseline period, for the early time period (0–15 s exposure) and for the late time period (45–60 s exposure). Flux was manually calculated. Velocity was determined using image analysis software (LabView, Department of Anesthesiology, University of Rochester) or video-streaming analysis software (MatLab, Department of Biomedical Engineering, Stony Book University).

Red blood cell flux, F cells/s, is calculated by F = (mt/p)/t, where mt is the number of fluorescent cells crossing a specified vessel plane in time, t, and p is the fraction of fluorescent cells in the total red blood cell population (0.4 ± 0.2%, mean ± SD). Individual velocities (µm/s), were measured as the distance traveled in one video field (1/60th s) for all fluorescent cells crossing the specified sampling plane during the selected period. The harmonic mean velocity, vc, was used as the estimate of bulk phase velocity (9). The apparent viscosity, {eta}app, was calculated from the relationship between vessel hematocrit, diameter (D) and the relative viscosity (37). The shear rate, {gamma} s–1, was calculated as: {gamma} = 8·vc/D, and used to calculate wall shear stress, T{omega} dyn/cm2, as: T{omega} = {eta}app·{gamma}.

Statistics.   The number of observations per group are reported in the figure legend or in the text. Comparisons between baseline and the early and late test period were calculated as: for diameter, {1+[(test – baseline)/(maximum – minimum)]}, where the maximum value was to topical 10–4 M adenosine and minimum value was to 10% oxygen gas, each used to confirm presence of tone. Flow values were calculated as [1+(test – baseline)/baseline]. Thus the changes are reported from unity, where 1 is the baseline value. Some comparisons are reported as the raw difference from baseline, calculated as: test – baseline. Values are reported as the means ± SD (for population parameters of Table 1) or means ± SE (for experimental comparisons) as indicated. Statistical analysis on repeated observations was determined between groups by ANOVA (repeated measures). For all statistics, differences were considered significant when P < 0.05 (45).


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  		Table 1. Hemodynamic characteristics of the observation site: entrance to the arteriolar network

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Table 1 provides the baseline characteristics of the observation site in arteriolar networks studied in the adult hamster cheek pouch tissue. The observation site was the entrance to an arteriolar network, defined previously (18). Remote vasoactive responses were obtained at the entrance of the network with stimulation (micropipette) at the terminal portion of the network. There was no significant difference between baseline values for the separate protocols.

Concentration dependence of the vitronectin receptor linked response.   In a prior study (19), the remote response to LM609 stimulation was shown to consist of two distinct hemodynamic phases. In the early phase, the upstream velocity increased within seconds; in the later phase, the upstream dilation began with an onset time of ~13 s and had reached a plateau by 45 s. In the present study, the data were analyzed in blocks of time corresponding to the early changes in velocity (e.g., peak in wall shear stress) occurring in the first 15 s after the onset of stimulation. The second block of time corresponded to the late changes between 45 and 60 s; the stimulus duration was 60 s. Figure 1 shows that both the peak wall shear stress prior to dilation and the peak dilation are a function of LM609 concentration. Furthermore, the fractional increase in diameter follows the same concentration dependence as the peak wall shear stress. Figure 1 also shows the recovered wall shear stress, that is, the upstream wall shear stress that is directly due to the dilation and associated hemodynamic changes in velocity and flux (i.e., the new steady state with LM609 exposure). Recovered wall shear stress is significantly less than baseline (unity) for the higher concentrations of LM609, indicating an overshoot in returning wall shear stress to baseline by the combination of hemodynamic changes. This agent stimulates a significant flow recruitment response, as described previously (Refs. 18, 24). The changes in red blood cell flux with each inhibitor agent are compiled in Table 2.


Figure 1
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  		Fig. 1. Shown is the concentration response relationship for the change in diameter and in wall shear stress (WSS) at the remote upstream location, as a function of downstream LM609 (0.5, 1, 5, 10 µg/ml). The change from unity reflects a fractional increase or decrease from baseline values. The peak WSS was the average during the first 15 s of stimulation; peak dilation was the plateau peak between 45–60 s of continued stimulation; recovered WSS at peak dilation was the average during the 45- to 60-s stimulation period (means ± SE, n = 30 animals, paired comparisons). *Larger than 0.5 µg/ml LM609; #less than unity.

 

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  		Table 2. Changes in red blood cell flux into the arteriolar network for each condition

 
Evaluation of KATP channel involvement.   Local dilation to abluminal stimulation through RGD (arginine-glycine-aspartate tri-peptide forming the recognition binding site for {alpha}vbeta3 to fibronectin, vitronectin, and other matrix proteins) binding sites has been shown to involve KATP channel-dependent hyperpolarization leading to inhibition of Ca influx (11, 31). Figure 2A shows that the remote dilation to pinacidil does not mimic the remote dilation to LM609. Whereas the remote dilation to pinacidil is concentration dependent, the changes in wall shear stress only occur after dilation, and predictably show a significant decrease only with greater dilation of the feed vessel. To evaluate whether KATP channels were linked to the remote dilation to LM609, we tested glybenclamide during 10 µg/ml LM609 stimulation. Figure 2B shows two key findings. First, although the peak increase in wall shear stress is attenuated by glybenclamide, it is not completely blocked even at 10–4 M. Second, KATP channel inhibition decreases the remote dilation by half when the peak wall shear stress stimulus is still significant, thus suggesting that blood velocity remains increased. DMSO (dimethylsulfoxide 0.01%, solvent concentration for 10–4 M glybenclamide) had no effect on the 10 µg/ml LM609 response, with the fractional increase in peak wall shear stress of 1.8 ± 0.3 and peak dilation of 1.6 ± 0.03 and recovered wall shear stress of 0.53 ± 0.11 (n = 6 animals). Glybenclamide alone had no effect on the baseline hemodynamic parameters of the observation site (changes from unity: peak WSS 1.06 ± 0.11; peak dilation 1.01 ± 0.08; recovered WSS 0.9 ± 0.1). Together, the data with pinacidil and glybenclamide are not consistent with KATP channel involvement per se in this response. To investigate whether the effects with glybenclamide were due to inhibition of hyperpolarization of the stimulus site, we tested 4-aminopyridine (4-AP; Fig. 2C). As with glybenclamide, the peak remote dilation was inhibited at lower concentrations of 4-AP and to a greater extent than was the peak increase in wall shear stress. 4-AP alone had no effect on the baseline hemodynamic parameters of the observation site (changes from unity: peak WSS 1.04 ± 0.09; peak dilation 1.08 ± 0.04; recovered WSS 1.04 ± 0.11). Thus hyperpolarization of the stimulus site appears to be involved in some component of the remote dilation that is not necessarily linked to the initial increase in velocity (e.g., peak wall shear stress response).


Figure 2
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  		Fig. 2. Shown is the concentration response relationship for the change in diameter and in wall shear stress (WSS) at the remote upstream location, as a function of 60 s downstream pinacidil (A) and as a function of glybenclamide (B) or 4-aminopyridine (4-AP; C) inhibition of 60 s 10 µg/ml LM609. Inhibitors were applied for 5 min prior to and during LM609 stimulation. The change from unity reflects a fractional increase or decrease from baseline values. The peak WSS was the average during the first 15 s of stimulation; peak dilation was the plateau peak between 45–60 s of continued stimulation; recovered WSS at peak dilation was the average during the 45- to 60-s stimulation period. Values are means ± SE. A: n = 14 networks in 7 animals; B: n = 18 networks in 16 animals; C: n = 6 networks in 6 animals; paired comparisons. *Differs from 10–8 M pinacidil (A) or LM609 alone (B, C).

 
Evaluation of chloride channel involvement.   The potential involvement of chloride channel activity (or depolarization) was suggested by the high concentration of glybenclamide required to diminish the response to LM609. Figure 3 shows the data with IAA-94, DIDS, and niflumic acid. Both IAA-94 and niflumic acid significantly blocked the early peak in wall shear stress, each in the micromolar range. DIDS attenuated the peak wall shear stress, but by 10–4 M DIDS, the response remained significant with respect to baseline wall shear stress. Together, this suggests that the early peak in wall shear stress requires depolarization at the stimulus site, perhaps via voltage-dependent chloride channels (see DISCUSSION). With DIDS, there was a gradual decrease in the remote dilation that accompanied the peak wall shear stress changes, with all responses remaining significant at the highest dosage. In contrast, with IAA-94 or niflumic acid, the peak remote dilation did not follow the same concentration dependence as the peak wall shear stress. With IAA-94 and with niflumic acid, a significant remote vasodilation remained at concentrations that completely blocked the peak wall shear stress. Thus a component of the LM609-stimulated remote dilation is not prevented by depolarization of the stimulated site. DIDS alone had no effect on the baseline hemodynamic parameters of the observation site (changes from unity: peak WSS 1.01 ± 0.06; peak dilation 1.14 ± 0.04; recovered WSS 0.49 ± 0.06). IAA-94 alone had no effect on the baseline hemodynamic parameters of the observation site (changes from unity: peak WSS 0.95 ± 0.09; peak dilation 1.07 ± 0.07; recovered WSS 0.98 ± 0.03). Niflumic acid alone had no effect on the baseline hemodynamic parameters of the observation site (changes from unity: peak WSS 0.96 ± 0.1; peak dilation 1.01 ± 0.08; recovered WSS 1.03 ± 0.08). The DIDS, IAA-94, and niflumic acid data together suggest that depolarization via chloride channels is involved in only the peak in wall shear stress (leading to flow-mediated dilation) and are selectively not involved in a parallel remote vasodilation mechanism. The effect of DIDS and niflumic acid on the LM609-stimulated remote vasodilation was compared with the effect of these chloride channel blockers on the well-characterized remote vasodilation to methacholine (10–4 M). Figure 4 shows that neither DIDS nor niflumic acid had a significant effect on the methacholine local or remote response, with the exception of 10–4 M DIDS. At this high concentration, DIDS attenuated both responses. Thus the effect of these chloride channel blockers to inhibit the LM609 response is not common to all remote vasodilatory pathways.


Figure 3
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  		Fig. 3. Shown is the concentration response relationship for the change in diameter and in WSS at the remote upstream location, as a function of IAA94 (indanyloxyacetic acid 94) (A), DIDS (B), or niflumic acid (C) inhibition of 60 s 10 µg/ml LM609. Inhibitors were applied for 5 min prior to and during LM609 stimulation. The change from unity reflects a fractional increase or decrease from baseline values. The peak WSS was the average during the first 15 s of stimulation; peak dilation was the plateau peak between 45–60 s of continued stimulation; recovered WSS at peak dilation was the average during the 45- to 60-s stimulation period. Values are means ± SE. A: n = 14 networks in 9 animals; B: n = 12 networks in 12 animals; C: n = 10 networks in 6 animals; paired comparisons. *Differs from with LM609 alone.

 

Figure 4
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  		Fig. 4. Shown is the concentration response relationship for the change in local and remote diameter as a function of DIDS (A) or niflumic acid (B) inhibition of 60 s methacholine (Mch), 10–4 M. Inhibitors were applied for 5 min before and during Mch stimulation. The change from unity reflects a fractional increase or decrease from baseline values. The local dilation was observed at the downstream stimulation site, and the remote dilation was observed upstream; dilations were the plateau peak ~15–20 s following the onset of continued stimulation. Values are means ± SE. A: n = 5 networks in 5 animals; B: n = 5 networks in 5 animals; paired comparisons. *Differs from with Mch alone.

 
Together, the potassium and chloride channel data with LM609 suggest that depolarization of the stimulus site is required for the early peak in wall shear stress (e.g., velocity increase) leading to upstream flow-mediated dilation, while hyperpolarization of the stimulus site is required for an independent remote vasodilation comprising approximately half of the total remote vasodilation.

Evaluation of tyrosine phosphorylation and protein kinase C involvement.   Several lines of published data by others suggest that potassium or chloride channel-mediated responses involve prior phosphorylation by PKC, possibly at a tyrosine residue. To evaluate tyrosine phosphorylation and PKC involvement in the LM609-linked response, genistein and chelerythrine were tested. Figure 5 shows that the remote dilation to 3.3 or to 10 µg/ml LM609 decreased significantly by 15-min exposure to 10 µM genistein. From Fig. 2, we note that a maximal remote response to LM609 is achieved with 10 µg/ml. Due to the concern that this saturating dose was affecting the apparent time course for genistein to inhibit the LM609 remote response, we tested the lower value (3.3 µg/ml). Each dose of LM609 showed the same time course for inhibition by genistein. The inactive analog, 10 µM daidzein (Fig. 5) did not affect the remote response to LM609. The vehicle for genistein and for diadzein was 0.1% ethanol. There is a transient remote constriction to 0.1% ethanol alone of –1.7 ± 0.6 µm, which dissipates by 5 min of continued application (at 5 min, 0.3 ± 0.5 µm) and does not return by 30 min of vehicle alone (at 30 min, –0.2 ± 0.5 µm). The remote dilation response to 10 µg/ml LM609 after 30 min 0.1% ethanol remains significant (5.2 ± 0.4 µm or 1.57 ± 0.4 change from unity). Genistein alone had no effect on the baseline hemodynamic parameters of the observation site (changes from unity: peak WSS 1.04 ± 0.12; peak dilation 1.09 ± 0.03; recovered WSS 0.98 ± 0.05). Likewise, with PKC inhibition, the remote dilation to 10 µg/ml LM609 was significantly blocked by 10–5 M chelerythrine (Fig. 6). To test the efficacy of the chelerythrine, we used 0.1 µg/ml phorbol 12-myristate 13-acetate (PMA), which alone induced a significant remote constriction of –2.8 ± 0.5 µm (–1.31 ± 0.05 change from unity), which was blocked with chelerythrine (0.32 ± 0.24 µm or 0.04 ± 0.02). Figure 6 shows the peak wall shear stress and peak diameter changes for LM609 stimulation with genistein, chelerythrine, or L-NNA. Genistein blocks all components to the LM609 response, indicating a central role of tyrosine phosphorylation. Chelerythrine inhibits the peak wall shear stress prior to dilation, but prevents only half of the remote dilation supporting a role for PKC stimulation in the velocity change, but not the secondary dilation response. L-NNA (applied upstream) does not significantly alter the LM609-stimulated increase in peak wall shear stress, but does block the remote dilation, reinforcing that half of the upstream remote dilation is NO-dependent flow-mediated dilation. Chelerythrine alone had no effect on the baseline hemodynamic parameters of the observation site (changes from unity: peak WSS 1.02 ± 0.07; peak dilation 1.09 ± 0.04; recovered WSS 0.92 ± 0.09). However, L-NNA alone induced a significant vasoconstriction (changes from unity: peak WSS 0.92 ± 0.05; peak dilation 0.75 ± 0.07; recovered WSS 0.85 ± 0.12).


Figure 5
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  		Fig. 5. Shown are remote dilation responses to 60-s stimulation by 3.3 or 10 µg/ml LM609. The remote dilations were tested at time = 0 min and then during simultaneous genistein (10 µM, n = 7) or daidzein (10 µM, n = 5), at 5, 15, and 30 min of exposure. Values are means ± SE. *Differs from response at time = 0.

 

Figure 6
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  		Fig. 6. Shown are the responses to 60-s stimulation with 10 µg/ml LM609 alone (combined for these data) or after 30 min 10–5 M genistein (n = 7) or after 5 min 10–5 M chelerythrine (n = 5) applied at the LM609 exposure site or after 5 min 10–4 M N{omega}-nitro-L-arginine (L-NNA; n = 6) applied at the upstream observation site. The change from unity reflects a fractional increase or decrease from baseline values. The peak WSS was the average during the first 15 s of stimulation; peak dilation was the plateau peak between 45–60 s of continued stimulation; recovered WSS at peak dilation was the average during the 45–60 s stimulation period. Values are means ± SE. *Differs from unity.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
This study shows that the flow recruitment response to {alpha}vbeta3 integrin (vitronectin receptor) ligation with LM609 requires tyrosine phosphorylation and involves protein kinase C activity. The data suggest that both a depolarizing and hyperpolarizing event occur at the stimulus site; the specific cell type harboring these changes was beyond the scope of this study; however, in a prior study, this LM609-elicited response required stimulation of a terminal arteriole that would dilate to nitroprusside (no response was seen with capillary stimulation) (17). The data support that localized ablumenal integrin binding on terminal arterioles directly induces network-specific changes in hemodynamic conditions, suggesting that dynamic interaction of the vessel wall with the surrounding matrix induces far-reaching changes in flow distribution.

The data with LM609 in the present study are summarized and interpreted as follows (Fig. 7). Inhibition of tyrosine kinase activity at the stimulus site prevents this entire integrin-mediated response, likely through inhibition of p125FAK (43), although possibly through inhibition of chloride channels (28). Depolarization (inhibition of chloride channels) at the stimulus site is essential for the initial remote increase in red blood cell velocity into the network. When the velocity increase is absent, the flow-mediated dilation does not occur. Likewise, inhibition of flow-mediated dilation at the observation site prevents a significant portion of the total dilation. It is clear that a secondary remote signal is simultaneously activated that transmits a remote vasodilation sufficient to increase flux into the network. Inhibition of protein kinase C at the stimulus site likewise blocks the initial velocity change, but not all of the secondary remote vasodilation. Remote vasodilation by muscarinic receptor stimulation does not occur by this pathway. Hyperpolarization (inhibition of potassium channels) is essential for the secondary dilation response, but does not affect the elevated velocity, nor the subsequent flow-mediated dilation. This response is not specific to KATP channels. The secondary dilation alone is sufficient to increase flow into the network. We speculate that the secondary dilation is transmitted through gap junctional signaling; this is supported by preliminary data (21). Both components to the response are required for full flow recruitment. Taken together, we suggest that downstream stimulation of the vitronectin receptor initiates an integrative flow recruitment mechanism with redundant means to increase upstream diameter and elevate flow into the stimulated arteriolar network. This is consistent with the action of this receptor during wound healing, in which elevated flow would provide a physiological stimulus for angiogenesis and means to amplify flow-limited solute transfer.


Figure 7
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  		Fig. 7. Schematic of proposed mechanism for vitronectin receptor stimulation of flow recruitment in the hamster cheek pouch microcirculation. Tyr-PO4, tyrosine phosphorylation; PKC, protein kinase C; VSM, vascular smooth muscle cell.

 
Vascular integrin receptors are integral membrane proteins involved in both the focal adhesion of the vessel wall to its matrix (abluminally; Refs. 4, 8) and in platelet and leukocyte interactions with the endothelium (luminally; Ref. 32). Luminal receptors are upregulated in atherosclerosis, primarily being found at sites of vascular injury (7, 49) and flow disturbance (25, 33); ligation of this receptor luminally blocks local flow-dependent dilation (33). The abluminal receptors are targeted in the present study and are activated by flow changes (38, 48, 50), induce local dilations (11, 31), are involved in vascular remodeling (8), and mediate permeability changes (38, 48, 50). Of importance here are the studies linking the receptor system to local dilation. Mogford et al.(31) showed that in isolated vessels, arginine-glycine-aspartate (RGD)-linked integrin stimulation induces a local dilation requiring chloride channel activity and is modulated by ATP-sensitive potassium channels. Our finding for remote dilatory responses differs from this local dilation in a number of ways. First, we demonstrate that remote from the local stimulation site there is first an elevated flow, and a portion of the remote dilation is flow dependent; the other portion of the dilation persists in the absence of an elevated wall shear stress. Second, although we also see a localized dilation with LM609 application, we know that merely a local dilation cannot induce this type of network flow recruitment based on prior work (19) and based on the data with pinacidil here. Thus we strongly suspect that with this one stimulation, we are initiating separate cellular responses, involving local dilation, and a network-wide remote response with redundant means to elevate flow. Third, the remote response consists of at least two mechanisms to decrease upstream resistance, and hence recruit a larger amount of flow. By examining the response within the intact flowing microvascular preparation, the present study contributes that vitronectin receptor stimulation is highly vasoactive, involving local and remote means to alter vascular resistance in a spatially patterned manner within a defined arteriolar network of the hamster cheek pouch tissue. Thus this hemodynamic mechanism represents a class of physiological responses in which the network response is not described solely by the local response, and instead represents a complex response requiring vascular communication, which leads to network or perhaps tissue-wide flow changes.

In a prior study we showed that stimulation of the abluminal vitronectin receptor induced a very specific type of flow recruitment, doubling inflow to the network with that flow traveling exclusively to the branch arteriole that was stimulated (17). In that study, LM609 induced a local dilation, when applied to terminal arterioles, the same network location as used here for this remote response. Physiologically this demonstrated a novel means for an arteriolar network to manipulate and recruit flow in a dynamic integrative manner. The present study adds that initiation of this response consists of two separate mechanistic pathways: one purely dilatory in nature and the other a more complex increase in flow leading to dilation. The physiological mechanism is thereby amplified by the redundant dilatory signal, and we speculate that the initial elevated velocity of flow (leading to the peak in WSS) serves two purposes. First, to increase solute delivery to a very specific vascular region where the receptor is stimulated and, second, to promote further chronic actions linked to this receptor system and to elevate flow in general. Our reasoning for this concerns the role of the abluminal {alpha}vbeta3 integrin (focal adhesion) receptor to bind vitronectin or fibronectin matrix components (4, 6). The emerging understanding suggests that permitted interactions of this focal adhesion receptor with the matrix are necessary for angiogenesis (6, 7). Additionally, one of the primary physiological stimuli for angiogenesis (capillary sprouting) is elevated flow within the microcirculation (24). We speculate that the elevated flow that accompanies/initiates angiogenesis (or then stimulates intimal hyperplasia in atherosclerotic disease) is due to activation of this integrin receptor in a manner as we have done experimentally. Interestingly, our prior work shows that stimulation of vascular endothelial cell growth factor receptor subtype 1 (VEGF-R1) initiates a similar integrative signal acutely within the intact flowing microcirculation (26). The similar components are specifically, the requirement for tyrosine phosphorylation to initiate the response and the upstream remote dilation requiring endogenous NO.

Whether the LM609 monoclonal antibody causes clustering of the {alpha}vbeta3 integrin receptors and the subsequent spontaneous phosphorylation and signal transduction is unknown. LM609 is well documented to prevent angiogenesis when used to chronically ligate this integrin receptor and prevent interaction of the sprouting endothelial cells with the extracellular matrix (6). Our acute data show that blocking either tyrosine phosphorylation or protein kinase C activity prevents the action of LM609 to stimulate flow upstream. The {alpha}vbeta3 integrin receptor system is linked to multiple physiological effects initiated by signal transduction events, including tyrosine phosphorylation of the p125FAK (focal adhesion kinase) and protein kinase C activity (49, 50). In the present study, genistein inhibition of the complete response supports the specific action of LM609 on the vitronectin receptor to mediate both components to the response. Potential downstream signal transduction events linked to this receptor are calcium-dependent chloride channels and ATP-sensitive potassium channels (35). The present study supports that both chloride and potassium channel activity is involved in the total physiological response by examining the responses in the intact flowing microvascular preparation.

The integrative response mechanism that we propose requires both a depolarization and hyperpolarization event at the stimulation site. We speculate that these events are occurring within different cell types; however, testing this was beyond the scope of the present study. There is a strong emerging precedence for independent action of endothelial and vascular smooth muscle cells in a variety of vascular responses (5, 14, 44). Part of the local response linked to RGD peptides outlined by Mogford et al. (35) show that KATP channel hyperpolarization with calcium sequestration occur within vascular smooth muscle. We therefore speculate that the hyperpolarizing events originate in vascular smooth muscle cells. The logical extension is that the proposed gap junction-linked ascending hyperpolarization signal must likewise occur along vascular smooth muscle cells, which is supported by experimental and theoretical evidence (5, 10, 14, 16).

How would depolarization, then, lead to an elevated flow velocity along the ascending flow path? Clearly more remains to be done to elucidate this mechanism. Vitronectin receptor stimulation of endothelial cells is known to cause depolarization that increases intracellular calcium and is directly linked to activation of the MAP kinase cascade and elevated transvascular permeability (2, 36). Consistent with this mechanism is our data showing that PKC is required for the initial velocity increase and not the hyperpolarization linked secondary dilation. We speculate that a potential mechanism to elevate flow prior to a change in arteriolar resistance is for this signaling pathway to increase hydraulic conductivity (permeability) within the downstream capillaries. Fluid extravasation could then lead to an increased dynamic pressure along the specified flow path and recruit flow exclusively to those capillaries. However, this mechanism would require that the vitronectin receptor on arteriolar endothelial cells is stimulated, not capillary endothelium. In a prior study, we showed that the remote vasodilatory response to LM609 only occurs when vascular smooth muscle cells are stimulated and not the capillary endothelium. In that study, we could not rule out an effect of vitronectin receptors on the endothelium of the terminal arterioles. Alternate mechanisms certainly cannot be ruled out, including nonspecific action of the agents on other endothelial systems.

In summary, this study contributes evidence that initiation of the LM609-induced remote response change requires tyrosine phosphorylation, protein kinase C activation, with evidence for both hyperpolarization and depolarization at the stimulation site. In the larger context of microvascular control, initiation of a programmed integrative response involving flow recruitment to an entire arteriolar network requires only that cellular stimulation occur locally at very small downstream regions of the network with the consequence that network flow is significantly increased and specifically directed to the stimulated flow path.


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This study was funded by National Heart, Lung, and Blood Institute Grant HL-55492 (to M. Frame).


   ACKNOWLEDGMENTS
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 ABSTRACT
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REFERENCES
 
Thanks to Patrick Valane, Judy Beckman, and Randall Fox for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. D. Frame, Dept. of Biomedical Engineering, Stony Brook Univ., HSC T18-030, Stony Brook, NY 11794-8181 (e-mail: mframe{at}notes.cc.sunysb.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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