Figure 1.
Fibro-vascular coupled morphology under confocal and TEM microscopy.
(A) Type V fibrocytes positive for S100 (green) abut capillary walls labeled by isolectin IB4 (blue). (B) Type V fibrocytes are positive for Na+/K+ ATPase β1 (red). (C) Type V fibrocytes also contain high levels of NO, as detected with the intracellular NO indicator, DAF-2DA (gray). (D) Magnification of panel B shows foot processes in contact with a capillary. (E) A multiple-foot process of a fibrocyte abuts a capillary wall. (F) A high magnification image shows a fibrocyte end-foot structure at the soma of a pericyte. The somas of the pericytes were labeled by an antibody for NG2, (red), and processes were labeled with an antibody for the structural protein, desmin (blue).) Capillary walls are labeled by phalloidin (green). (G) and (H) Fibrocytes contact capillaries with enlarged endings. (I) The endings display electron-dense membrane regions rich in mitochondria. Abbreviations: FC, fibrocyte; EC, endothelial cells; PC, pericyte; Mt, mitochondria. Calibration bars in H and I are 500 nm.
Figure 2.
Photolysis of caged Ca2+ in fibrocytes initiates a propagating Ca2 wave in capillaries.
(A) Fibrocytes communicate with nearby vascular cells. The fibrocyte was stimulated by photolysis at 1 (the purple arrow indicates the site of the uncaging flash). Note the photolysis-evoked Ca2+ wave (1 FC) propagates sequentially to vascular cells [2 (PC), 3 (PC), 4 (PC), and 5 (EC)]. (B) Ca2+ probe fluorescence from stimulation of fibrocytes propagates with delay to vascular cells.
Figure 3.
Photolysis of caged Ca2+ in fibrocytes evokes vasodilation in vivo.
((A) Photolysis-evoked vasodilation (left, before photolysis; right, after photolysis; white dotted lines in A, B indicate sites of dilation). (B) Photolysis-evoked, time-dependent change in intracellular Ca2+ in stimulated fibrocyte (green line) correlates with the change in capillary diameter (red line). (C) Mean fluorescent signal of the Ca2+ indicator is significantly increased. (D) Mean capillary diameter is significantly increased (n = 8, P<0.01).
Figure 4.
Photolysis of caged Ca2+ in fibrocytes evokes vasodiation in vivo through COX-1 signaling.
(A) mRNA for Cox-1 and Cox-3 is expressed in the cochlear lateral wall (left). COX-1 protein is selectively expressed in type V fibrocytes, but not in vascular cells (right). (B) Photolysis evokes vasodilation (left, before photolysis; right, after photolysis; white dotted lines in left, right indicates sites of dilation). (C) Lack of photolysis-evoked vasodilation is shown (left, before photolysis; right, after photolysis; white dotted lines in left, right indicates sites of changes of capillary diameter). (D) Mean capillary diameter is significantly increased before and after photolysis (n = 8, P<0.01). In contrast, mean capillary diameter is unchanged in tissues treated with a COX-1 inhibitor (n = 10, P>0.05).
Figure 5.
Sound-induced changes of intracellular Ca2+ in fibrocytes, blood-flow velocity, and capillary diameter.
(A) and (B) Changes in cochlear blood flow velocity and capillary diameter under a variety of conditions: control, sound stimulated, COX-1 inhibited, and sound stimulated with COX-1 and CBX inhibition. Sound stimulation alone caused significant increases in capillary diameter and blood-flow velocity (n = 15, P<0.05). However, prior perfusion of the vessel-window with the COX-1-specific inhibitor, SC 560, or with the gap junction blocker, CBX , essentially blocked the sound-induced dilation. The cartoon shows the sound-stimulation protocol. (C) Intracellular Ca2+ signals are shown under control (left, no sound) and sound-stimulated conditions (middle, sound on). Fluorescence of the intracellular Ca2+ probe in some fibrocytes (arrows) returns to normal about 2 min after sound stimulation (right, sound off). (D) Mean Ca2+ signal was significantly higher in the sound stimulated fibrocytes.
Figure 6.
A working model of fibro-vascular coupled signaling in the inner ear.
The schematic diagram illustrates selected aspects of fibrocyte signaling. Sound stimulation (red dotted line) activates hair cells and initiates Ca2+ signaling in fibrocytes. While the gating mechanisms for the Ca2+ signaling have not been determined, mechanical vibration or metabolic activity, such as K+ recycling, initiated by sound might underlie the gating. COX-1 is regulated by the elevation of Ca2+ in fibrocytes. The COX-1 converts arachidonic acid into metabolic intermediates, including PGE2, which diffuse into the perivascular space to elicit vasodilatation.