Discussion
The data presented here is the first to investigate the role of SVEP1
and integrin α9β1 in vasoconstriction. SVEP1 was found to bind to
integrin α9β1 and for the first time, the closely related integrin α4β1.
Cell adhesion studies suggest that SVEP1 binds to integrin α4β1 through
its CCP21 domain. Within the vasculature and in isolated VSMCs, we found
expression of SVEP1, integrin α4β1, and α9β1 to be predominantly
localised within the media layer confirming previous
data23. Reduction in SVEP1 levels led to an
increase in Ca2+ release in response to a panel of
vasoconstrictors in iVSMC. Similar maximal increases in
Ca2+ levels were seen upon inhibition of integrin α4β1
or α9β1, however, no additional alterations in Ca2+release were seen upon co-inhibition of SVEP1 and the integrins.
Comparable results were detected in whole vessel contraction withSvep1+/- aortic rings displaying enhanced
contraction compared to aortas from litter-mate controls, whilst
inhibition of integrin α4β1 or α9β1 also enhanced contraction. As seen
in isolated cells, no additional contraction was seen when both SVEP1
and integrin α4β1 or α9β1 were simultaneously inhibited, suggesting a
level of redundancy or a ceiling effect of SVEP1 regulation upon vessel
contraction.
While no vasoregulatory role has previously been described for SVEP1, a
comparable study within the airway found ligation of integrin α9β1 to
prevent airway hyperresponsivness8, a phenotype
similar to the role of SVEP1 reducing vascular hyperresponsivness
presented here. While the role of SVEP1 was not specifically
investigated in the airway study, GTEx and the Human Protein Atlas
describe SVEP1 expression in the lung, oesophagus and intestine,
providing the possibility that SVEP1 has a similar role in reducing
smooth muscle hyper-contractility in several tissues.
Our studies showed PKC-mediated influx of Ca2+ via
VGCCs to be the dominant mechanism in U46619-mediated vessel
contraction. U46619 caused the most reliable aortic vessel contractions
in studies, however, the signalling mechanisms downstream of the
receptor are varied across smooth muscle tissue types: In the intrarenal
artery, the mechanism is VGCC dependent, and partially PKC
dependent31 as seen within our aortic model. Within
the bovine pulmonary artery contraction does not require
VGCCs32, whilst in the rat mesenteric
artery33 and the rat caudal artery34PKC is not required, suggesting a diverse downstream signalling
mechanism across tissues. Our own studies demonstrate the importance of
a PKC-dependent VGCCs signalling mechanism in aortic smooth muscle (Fig.
7). There is evidence that other GPCRs can activate integrins via
PKC-dependent “inside-out signalling”35,36, with PKC
activity upstream of integrins the activated integrin inducing tyrosine
phosphorylation of focal adhesion-associated
proteins35-37. Studies into VSMC contraction initiated
by direct integrin ligation also indicated a dominant role for VGCCs.
Whole-cell recordings from arteriolar myocytes show αvβ3 to inhibit VGCC
current3,5. Integrin α4β1 and α5β1 ligation enhances
L-type Ca2+ current 4-7, and
integrin α7β1 regulates both transient elevation of
[Ca2+]i from IP3evoked Ca2+ release from intracellular stores and
extracellular Ca2+ influx through VGCC in skeletal
muscle38. These described studies administer synthetic
peptides such as RGD or LDV to mimic important vasoactive ECM fragments,
termed matrikines, which are otherwise un-exposed within the full-length
ECM molecules39-41. Dysregulation of the ECM is linked
to several vascular-associated diseases including
CAD42-44, heart failure45, and
stroke46. SVEP1 is known to be targeted by the
protease ADAMTS-7 47 which is linked to
atherosclerotic plaque formation48-50, and contains
the linear peptide sequences RGD (which binds to integrin αv proteins)
and LDV sequences (which bind to integrin α4 and
α9)51-53. It would be interesting to determine whether
SVEP1 breakdown products had altered vasoregulatory effects compared to
full length SVEP1.
Our data identifies SVEP1 and integrins α4β1 and α9β1 as new mediators
of GPCR-mediated vasoconstriction. Notably, human genetic studies have
identified associations between variants in both SVEP1 and integrin α9β1
and BP. Our data provide a possible explanation for both associations
and warrants further investigation. Future studies should investigate
whether similar contractile profiles are seen within resistance arteries
and whether these effects contribute to an altered BP and if this is
affected by the BP-associated variants.
In conclusion we have described for the first time how the ECM protein
SVEP1 prevents VSMC hyper-contractility via integrin α4β1 and/or α9β1 by
reducing Ca2+ influx through VGCCs, providing a new
link between a novel ECM protein and VSMC contraction.