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Ahmed et al. Vessel Plus 2018;2:36 I http://dx.doi.org/10.20517/2574-1209.2018.51 Page 5 of 13
the sarcolemma, which causes opening of the calcium channels and subsequent depletion of calcium
intracellular store.

After an increase of cytoplasmic calcium concentration has been established, calmodulin becomes bound by
4 calcium ions. The resulting calcium-calmodulin complex interacts with and activates myosin light chain
[43]
kinase (MLCK) . Next, MLCK phosphorylates MLC-20 (also known as the regulatory light chain) on the
serine-19 and threonine-18 residues. The phosphorylation of serine-19 causes a resulting increase in the
2+
activity of the Mg -ATPase and this effect is further enhanced by the phosphorylation of the latter residue.
From this, the cross-bridge cycling is initiated and the myosin head can actively pull on the thin filament of
[44]
the actin to induce contraction of the muscle .
Rho/Rho-associated protein kinase pathway
In the absence of external contractile stimuli, the MLC-20 light chain remains phosphorylated at a low level.
[36]
This low level leads to a slower tonic form of contraction, which regulates the vascular tone . A calcium-
independent pathway that involves Rho/Rho-associated protein kinase (ROCK) signalling regulates VSMC
tonic contractions [Figure 2]. This pathway not only caters to contractile function, but also extends to
[45]
smooth muscle cell migration, proliferation and apoptosis . RhoA, part of the Ras superfamily, is a GTPase
[46]
which can act as a molecular switch between a GTP/GDP bound state . In resting conditions, the Rho GDP
dissociation inhibitor targets GDP-Rho for binding, as a means to localise the GTPase from the membrane
to the cytosol. However, activation of GPCR receptors, in particular Ga12/13 subtypes, can catalyse GTP for
GDP exchange in RhoA by binding to p115 RhoGTPase guanine nucleotide exchange factors . In its GTP
[47]
bound form, RhoA can interact with target proteins by utilising its C-terminal geranyl-geranylated tail to
[47]
anchor itself to the plasma membrane .
One of the target proteins activated by RhoA is ROCK . ROCK is a member of the protein kinase A, G
[48]
and C family of protein kinases, and is characterised as a serine/threonine kinase. There are two isoforms
[49]
of this kinase, referred to as ROCK1 and ROCK2, with expression of both present in VSMCs . Its
structure is composed of an N-terminal kinase domain, a central coiled-coil domain and a C-terminal
pleckstrin homology domain that associates with the Rho GTPase . ROCK has many effects within
[47]
VSMCs and influences actomyosin activity by two main pathways. Firstly, ROCK actively regulates
cytoskeletal organisation by preventing actin filament depolymerisation . Secondly, ROCK inhibits
[48]
myosin light chain phosphatase (MLCP). MLCP has a structure that is composed of three subunits; a 37
[36]
kDa catalytic subunit, a variable subunit and a myosin-binding subunit . The myosin-binding site is
crucial for its regulation and is subject to phosphorylation, specifically at residues. Threonine-695/697 (major
site), serine-849/854 and threonine-850/855 [47,50] . Phosphorylation prevents MLCP from regulating the MLC
phosphorylation state and increases the basal phosphorylated MLC level, stimulating VSMC contraction
[50]
and augmenting vascular tone .

MEMBRANE ANCHORS TO THE ACTIN CYTOSKELETON
VSMCs make connective junctions to their surrounding environment, which includes the ECM and
neighboring cells within the vasculature. These adhesions play a vital role in determining morphology
and VSMC function. The adhesion molecules that are utilised by VSMCs can be separated, despite their
[51]
structural and functional similarities .

Cell-cell adhesions
Cadherins are the primary molecules in cell-cell adhesion formation. The most abundant isoforms are E
(epithelial)-, P (placental)- and N (neuronal)-cadherins, all of which belong to the type I classical cadherin
family [52,53] . N-cadherin is the predominant cadherin in VSMCs and mediates cell-cell adhesion formation
[53]
with neighbouring endothelial cells as well as other VSMCs . The N-cadherin adhesion plays important roles