Angiotensin II

12/1/2023 1
 RAS- Angiotensin II
• Tigerstedt and Bergmann first published data in 1898
on a substance secreted by renal cortex which is able
to increase arterial pressure when injected- Renin
• The signaling events elicited in vascular smooth muscle
cells by angiotensin II (Ang II) were considered to be
– rapid, short-lived, and divided into separate linear
pathways
• Where intracellular targets of the PLC-C/DAG-Ca2+ axis
were distinct from those of the TK- and MAPK
dependent pathways.
12/1/2023 2
 Angiotensin II
• Downstream signals- elicit complex and multiple
responses.
• Ang II induces a multitude of actions in:
– various tissues,
– signaling events following occupancy and activation of
angiotensin receptors are tightly controlled and extremely
complex.
• Alterations of these highly regulated signaling
pathways in vascular smooth cells is pivotal in
structural and functional abnormalities that underlie
vascular pathological processes in cardiovascular
diseases such as hypertension, atherosclerosis

12/1/2023 3
 Angiotensin II
• The vascular wall is an active, pliable (flexible)
and integrated organ made up of cellular
(endothelial cells, vascular smooth muscle
cells, and fibroblasts) and noncellular
(extracellular matrix) components.
• It is not a static organ; the components
dynamically change shape, increase, decrease,
or reorganize, in response to physiological and
pathological stimuli

12/1/2023 4
 Angiotensin II
• In the intact arterial media, smooth muscle
cells and matrix are responsible for:
– structural and functional characteristics of the
vessel wall, including contraction-relaxation,
– growth, development, remodeling, and repair, and
for the pathogenesis of cardiovascular disease,
such as atherosclerosis and hypertension

12/1/2023 5
 Angiotensin II
• Many local and systemic factors regulate
vascular smooth muscle cell function,
including vasoactive peptides, such as
– Ang II and endothelin-1 (ET-1), that stimulate
vasoconstriction and growth
– Vasorelaxing factors, such as nitric oxide,
prostacyclin, and C-type natriuretic peptide that
induce vasodilatation by increasing levels of cyclic
nucleotides

12/1/2023 6
 Ang II
• Numerous actions on vascular smooth muscle—it
modulates
– Vasomotor tone,
– it regulates cell growth and apoptosis,
– It influences cell migration and extracellular matrix
deposition,
– it is pro-inflammatory, and it stimulates production of
other growth factors [e.g., platelet-derived growth factor
(PDGF)] and vasoconstrictors (e.g., ET-1).
• Ang II plays a fundamental role in controlling:
– the functional and structural integrity of the arterial wall
– regulating blood pressure and in pathological mechanisms
underlying vascular diseases.
12/1/2023 7
 Ang II
• Multiple actions of Ang II are mediated via
specific, highly complex intracellular signaling
pathways

• Includes the interconnected molecular cascades

that transmit information from the cell
membrane receptor to the intracellular proteins
that regulate cell activities such as
– contraction, cell growth, mitogenesis, apoptosis,
differentiation, migration, and other specialized
functions.

12/1/2023 8
 The Renin Angiotensin System
• Ang II, an octapeptide hormone, is the active
component of the renin-angiotensin system (RAS).
• It regulates blood pressure, plasma volume via
aldosterone-regulated sodium excretion,
sympathetic nervous activity, and thirst responses.
• It also plays a fundamental role in pathological
adaptation, as manifested in myocardial
remodeling after myocardial infarction and in
vascular remodeling in hypertension.

12/1/2023 9
 Juxtaglomerular apparatus (JGA)

12/1/2023 10
 Juxtaglomerular apparatus (JGA)
• A specialized collection of two cell types, macula
densa cells and juxtaglomerular cells, located at the
juncture of the afferent and efferent arterioles with a
portion of the distal convoluted tubule of the
nephron of the kidney;
• Two cell types participate in the negative feedback
regulation of
– systemic blood pressure and blood volume via the renin-
angiotensin system,
– local control of glomerular filtration rate (GFR)

12/1/2023 11
 GFR
• GFR - glomerular filtration rate is the best test to
measure level of kidney function and determine stage of
kidney disease.
• Calculated from the results of blood creatinine test, age,
body size and gender.
• If GFR is low, kidneys are not working as well as they
should.
• The earlier kidney disease is detected, the better the
chance of slowing or stopping its progression
GFR
 The Renin Angiotensin System
• Ang II is produced systemically via the classical
or renal RAS, and locally via tissue RAS.
• Circulating renal-derived renin cleaves
hepatic-derived angiotensinogen at the N
terminus to form the decapeptide,
angiotensin I,
– which is converted by the dipeptidyl
carboxypeptidase, angiotensin-converting enzyme
(ACE), in the lungs, to the active Ang II
12/1/2023 15
 The Renin Angiotensin System
• Ang I can also be processed into the
heptapeptide Ang-(1-7) by three tissue
endopeptidases, neutral endopeptidase (NEP)
24.11, NEP 24.15, and NEP 24.26
• Ang II is degraded by aminopeptidases to Ang
III and Ang IV

12/1/2023 16
 Chymase
• Non-ACE pathways, which could be particularly
important in pathological states, have been
demonstrated.
• Chymotrypsin-like serine protease (chymase) may
represent an important pathway for conversion
of Ang I to Ang II in the human heart and kidney.
• Functional chymase pathway have also been
demonstrated in human vascular tissue and in
dog carotid artery

12/1/2023 17
Classical The Renin Angiotensin System

12/1/2023 18
Counter- regulatory renin–angiotensin
Counter- regulatory renin–angiotensin
non- canonical axis
of the renin–
angiotensin system
consists of
angiotensin
1–7 , angiotensin 1–
9, angiotensin-
converting enzyme
2, the type 2
angiotensin II
receptor (AT2R),
the proto- oncogene
Mas receptor and
the Mas- related G
protein- coupled
receptor member D.
aspartate
decarboxylase (AD)
aminopeptidase A (APA)
aminopeptidase N (APN)
Signal transduction mechanisms of the counter-
regulatory RAS
 Angiotensin-converting enzyme 2
• ACE2 is a monocarboxypeptidase that degrades
angiotensin II with high efficiency leading to the
formation of angiotensin-(1–7)
• ACE2 within the kidneys is largely localized in tubular
epithelial cells and in glomerular epithelial cells.
• Decreased glomerular expression of this enzyme
coupled with increased expression of ACE has been
described in diabetic kidney disease, both in mice
and humans with type 2 diabetes.

12/1/2023 22
 Angiotensin-converting enzyme 2

12/1/2023 23
 Angiotensin-converting enzyme 2
• ACE2 genetic ablation and pharmacological ACE2
inhibition have been shown to increase albuminuria
and promote glomerular injury.
• ACE2 have shown the ability to rapidly metabolize
Ang II in vivo and form the basis for future studies to
examine the potential of ACE2 amplification in the
therapy of diabetic kidney disease and cardiovascular
disease

12/1/2023 24
 ACE2 AS A THERAPEUTIC TARGET

• The therapeutic potential of amplifying ACE2 activity is

worthy of intensive investigation either as a primary therapy
within the RAS or as a complement to existing ones
• ACE2 deficiency has been associated with enhanced
expression of inflammatory markers, increased oxidative
stress, and may be proatherogenic
• Many of these actions are likely related to the local
accumulation of Ang II.
• Much of the expected therapeutic benefit of ACE2
amplification stems from its ability to lower the levels of Ang II
while concomitantly increasing the formation of Ang-(1–7).
 ACE2 AS A THERAPEUTIC TARGET
• An interesting study in which STZ-induced diabetic rats
were given either the ACE inhibitor benazepril, an
adenoviral (Ad)-ACE2 injection, or both for 4 weeks.
• Their results suggested that although both ACE inhibition
and ACE2 over expression help improve signs of diabetic
nephropathy, one was not more effective than the other.
• There is a need for much more evidence showing that ACE2
amplification in itself can provide organ protection beyond
what is already achieved by current blockers of the RAS.
• One question that needs to be addressed is the role of
ACE2 in the face of blockade of the RAS such as ACE
inhibition or AT1 receptor blockade.
 Diminazene aceturate

• Diminazene aceturate (DIZE) is a US FDA approved small

molecule which belongs to the group of aromatic diamidines
used against babesiosis [malaria-like parasitic disease] and
trypanosomiasis
• It has also been shown to exert an “off target” ACE2
activating effect
 Diminazene Aceturate
• It significantly attenuated the myocardial infarction induced
decrease in fractional shortening, improved the maximal rate
of rise of left ventricular pressure (LVP) and reversed
ventricular hypertrophy
• It prevented the development of pulmonary hypertension
(PH) induced in male Sprague Dawley rats
Diminazene Aceturate

DIZE: Diminazene Aceturate , LD: low dose (5 mg/kg/day), HD: high dose (15
mg/kg/day), 4 weeks.

Diminazene Aceturate prevents nephropathy

Goru et al, British Journal of Pharmacology (2017), 174, 3118–3130
BITS Pilani, Pilani Campus
Diminazene Aceturate

All the values are represented as mean ± S.E.M.; n = 6 rats per group; *P < 0.05 vs NC, @P<0.05 vs DC and #P<0.05 vs
DC+LD. NC (normal control), DC (diabetic control), LD (low dose of DIZE (5mg kg-1)) and HD (high dose of DIZE (15mg kg-
1).
DIZE inhibited diabetes induced renal fibrosis and apoptosis
through increasing Glomerular ACE2 and AT2 protein expression.
BITS Pilani, Pilani Campus
Diminazene Aceturate

Mechanism of DIZE
BITS Pilani, Pilani Campus
 Tissue RAS
• The RAS was originally regarded as a
circulating system.
• Many of its components are localized in
tissues indicating the existence of a local
tissue RAS as well

12/1/2023 32
 Angiotensin Converting Enzyme (ACE)
• ACE exists in plasma (as the circulating
hormone), in the interstitium and
intracellularly.
• Tissue ACE is present in all major organs,
heart, brain, blood vessels, adrenals, kidney,
liver, and reproductive organs, and is already
functional in utero.
• Tissue ACE activity seems to peak during the
phase of major organ development

12/1/2023 33
 ACE
• All components of the RAS, except renin, have
been demonstrated to be produced in the
vasculature.
• ACE is found in high concentrations in the
adventitia, as well as in cultured vascular smooth
muscle and endothelial cells
• Since vascular renin is absent, local generation of
Ang II in the interstitium is regulated by tissue
ACE that is probably dependent on circulating
renin
12/1/2023 34
 Ang II

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12/1/2023 36
 Angiotensin Receptors
• In mammalian cells, Ang II mediates its effects
via at least two high-affinity plasma
membrane receptors, AT1 and AT2.
• Both receptor subtypes have been cloned and
pharmacologically characterized
• Two other Ang receptors have been described,
AT3 and AT4 subtypes.

12/1/2023 37
 AT3R
• The AT3 receptor subtype, initially described
in the neuro 2A neuroblastoma cell line is
peptide-specific recognizing mainly Ang II.
• This subtype does not bind to nonpeptide
ligands such as losartan (selective AT1
receptor antagonist) or PD123319 (selective
AT2 receptor antagonist), and has only been
observed in cell lines.

12/1/2023 38
 AT4R
• AT4 receptor, which is distributed in heart, lung,
kidney, brain, and liver, binds Ang IV but not
losartan or PD123319.
• Pharmacology of AT3 and AT4 receptors has not
been fully characterized.
• Not yet included in a definitive classification of
mammalian AT receptors as defined by the
International Union of Pharmacology
Nomenclature Subcommittee for Angiotensin
Receptors.
12/1/2023 39
 Pharmacology of angiotensin-R

12/1/2023 40
 Characteristics of AT1 and AT2 R

12/1/2023 41
Signalling pathways for AT2 receptor
AT2 Receptor Agonist
C21
 Angiotensin II-Dependent Signaling Pathways

• Ang II elicits complex highly regulated

cascades of intracellular signal transduction
that lead to short-term vascular effects, such
as contraction, and to long-term biological
effects, such as cell growth, migration,
extracellular matrix deposition, and
inflammation.

12/1/2023 46
 Angiotensin II-Dependent Signaling Pathways

• Ligand receptor binding on the external cell

membrane surface induces the interaction
between the receptor and effector protein on
the internal cell membrane surface via G
proteins (heterotrimeric proteins comprised of
α β,and ɣ subunits).

12/1/2023 47
 Angiotensin II-Dependent Signaling Pathways

• The signaling processes are multiphasic with

distinct temporal characteristics
• Immediate, early, and late signaling events
occur within seconds, minutes, and hours,
respectively

12/1/2023 48
 Angiotensin II-Dependent Signaling Pathways

• Ang II-induced phospholipase C (PLC) phosphorylation

and Src activation occur within seconds and constitute
immediate signaling events,
• Activation of phospholipase A2 (PLA2), phospholipase
D (PLD), tyrosine kinases and mitogen-activated
protein kinases (MAPKs) occurs within minutes and are
early signaling processes,
• Generation of oxidative stress and protein synthesis,
which occur within hours, make up late signaling
events.

12/1/2023 49
 Angiotensin II-Dependent Signaling Pathways

12/1/2023 50
 Immediate Signaling
• Ang II-elicited vascular contraction is rapid and utilizes various
signaling mechanisms that occur within seconds of Ang II binding to
its receptor.
• These immediate signal transduction processes include:
– G protein mediated activation of PLC, leading to phosphatidylinositol
hydrolysis and formation of inositol trisphosphate (IP3) and
diacylglycerol accumulation (DAG);
– Increase in cytosolic free calcium concentration ([Ca2+]i) by increasing
Ca2+ influx and mobilizing intracellular Ca2+;
– activation of protein kinase C (PKC);
– changes in intracellular pH (alkalinization) via stimulation of the
Na+/H+ exchanger;
– changes in intracellular free concentrations of Na+ ([Na+]i) and Mg2+
([Mg2+]i);
– activation of the Src family of kinases

12/1/2023 51
 Immediate Signaling

12/1/2023 52
 Events
• Stimulation of Phospholipase C and
Phosphatidylinositol Hydrolysis:
– rapid, PLC-dependent hydrolysis of
phosphatidylinositol-4,5-bisphosphate (PtdInsP2)
to yield water soluble IP3 and membrane bound
DAG
– PLC is a family of at least three related genes: PLC-
β, PLC-γ, and PLC-δ

12/1/2023 53
 Events
• Increased Intracellular Free Calcium Concentration.
– Ang II-stimulated Ca2+ signaling is complex and occurs via
multiple pathways to elicit an integrated Ca2+ signal.
– Ang II typically mediates a biphasic [Ca2+ ]I response
comprising a rapid initial transient phase and a sustained
plateau phase
– The first transient [Ca2+ ]I is generated primarily by IP -
3

induced mobilization of intracellular Ca2+

– second [Ca2+ ] phase, which appears to contribute to the
I

sustained Ang II-induced vasoconstriction, is dependent on

external Ca2+ and is the result of transmembrane Ca2+ influx
– Voltage dependent calcium channels (VDCC)

12/1/2023 54
 Calcium entry
• When calcium signaling is stimulated in a cell, Ca2+
enters the cytoplasm from one of two general sources:
– it is released from intracellular stores, or it enters the cell
across the plasma membrane.
– Both processes often occur either simultaneously or
sequentially.
• In many excitable cells (neuronal cell), entry of Ca2+ can
be activated by membrane depolarization.
• This signal is then amplified as the entering Ca2+
triggers additional mobilization of intracellular Ca2+
stores through activation of Ryanodine Receptor (RyRs)
channels, a process known as Ca2+ induced Ca2+ release
(CICR).
12/1/2023 55
 Calcium entry
• Non-excitable cells, an important initiating step is
intracellular release of Ca2+ from internal stores by
binding of a second messenger to its receptor in the
endoplasmic reticulum (ER).
• Commonly, this messenger is inositol 1,4,5-
trisphosphate (IP3)
• Ca2+ entry can be signaled by a variety of processes,
including direct activation by surface receptors and
activation by a variety of second messengers.
• Most commonly observed mechanism of regulated
Ca2+ entry in non-excitable cells is a process know as
capacitative Ca2+ entry or store-operated Ca2+ entry

12/1/2023 56
 Capacitative calcium entry
• Capacitative Ca2+ entry involves the regulation of
plasma membrane Ca2+ channels by the filling
state of intracellular Ca2+ stores in the
endoplasmic reticulum (ER).
• Agonist activation results in the production of
Ins(1,4,5)P3, which results in discharge of stored
Ca2+.
• Alternatively, Ca2+ stores can be discharged
passively through the use of reagents such as
thapsigargin
12/1/2023 57
 Thapsigargin
• It is non-competitive inhibitor of a class of enzymes
known by the acronym SERCA, which stands for sarco/
endoplasmic reticulum Ca2+ ATPase.
• It raises cytosolic (intracellular) calcium concentration
by blocking the ability of the cell to pump calcium into
the sarcoplasmic and endoplasmic reticula which
causes these stores to become depleted.
• Store-depletion can secondarily activate plasma
membrane calcium channels, allowing an influx of
calcium into the cytosol.

12/1/2023 58
 Proposed Mechanism

(A) A diffusible signal – Ca2+-influx (B) Exocytosis model:

factor (CIF) – Is released from the Depletion of stores causes fusion of vesicles
ER and activates plasma membrane containing CRAC channels with the plasma
store-operated channels. membrane.

12/1/2023 59
 Proposed Mechanism
(C) In the Ca2+ regulation:
Ca2+ discharged from a replete Ca2+ pool
keeps the channels in an inhibited state.
Discharge of the stores removes the
source of this inhibitory Ca2+ and relieves
the inhibition.
12/1/2023
(D) In the conformational coupling:
Discharge of Ca2+stores leads to a
conformational change in the Ins(1,4,5)P3
receptor, which is transmitted to plasma
membrane Ca2+channels by a direct protein-
protein interaction.
60
 Events
• Activation of Protein Kinase C
– a serine/threonine kinase that is a member of a
multigene family consisting of at least 11 isoenzymes
– Ang II stimulates the translocation of cytosolic PKC to
the plasma membrane where the activated enzyme
phosphorylates specific proteins associated with
vascular function
– PKC is implicated in Ang II-induced vascular
contraction as well as in vascular smooth muscle cell
growth

12/1/2023 61
 Events
• Activation of Protein Kinase C
– effects are mediated via activation of the Na+/H+
exchanger leading to intracellular alkalinization, an
important modulator of actin-myosin interaction, and
of contraction
– PKC induces its actions through phosphorylation of
tyrosine kinases, such as proline-rich tyrosine kinase
(PYK2), p130Cas and Src family tyrosine kinases, and by
stimulating MAP kinase signaling pathways
– The PKC isoform that activates ERK-1 and ERK-2
(extracellular signal regulated kinases) in vascular
smooth muscle cells has been identified as PKC-z.

12/1/2023 62
 Src Kinase
• Src (pronounced "sarc" as it is short for sarcoma) is a
proto-oncogene encoding a tyrosine kinase originally
discovered by J. Michael Bishop and Harold E. Varmus,
for which they were awarded the 1989 Nobel Prize in
Physiology or Medicine.
• It belongs to a family of non-receptor tyrosine kinases
called Src family kinases.
• This gene is similar to the v-Src gene of Rous sarcoma
virus.
• This proto-oncogene may play a role in the regulation
of embryonic development and cell growth.
 Src Kinase
• Src family kinase family includes nine members: Src,
Yes, Fyn, and Fgr, forming the SrcA subfamily, Lck,
Hck, Blk, and Lyn in the SrcB subfamily, and Frk in its
own subfamily.
• Frk has homologs in invertebrates such as flies and
worms, and Src homologs exist in organisms as
diverse as unicellular choanoflagellates, but the SrcA
and SrcB subfamilies are specific to vertebrates
 Events
• Activation of Src Family Kinases:
– The Src family of protein tyrosine kinases that
characteristically interact with transmembrane
tyrosine kinase receptors, also interact
functionally with G protein-coupled receptors
such as AT1
– 14 Src-related kinases have been identified, of
which the 60-kDa c-Src is the best characterized
– Src plays an important role in Ang II-induced
phosphorylation of PLC-γ and IP formation.
3

12/1/2023 65
 Events
• Angiotensin II Increases Intracellular Free
Concentrations of Na+ and Decreases Intracellular
Free Concentrations of Mg2+
– Ang II raises [Na+]i and reduces [Mg2+]i in a concentration-
dependent fashion in vascular smooth muscle cells
– These effects are rapid and maximal responses occur
within 40 to 60 s
– Ang II-stimulated increase in [Na+]i and reduction in
[Mg2+]i influence vascular smooth muscle contraction
directly or indirectly by modulating [Ca2+]I
– The cellular mechanisms regulating [Mg2+]i are unknown

12/1/2023 66
 Early Signaling

• Multiple intracellular transduction pathways

that are linked to long term regulation of
vascular smooth muscle cell function, such as
growth, migration, deposition of extracellular
matrix, and production of growth factors.
 Early Signaling

• These processes are initiated by signaling

pathways that are stimulated by Ang II within
minutes and include:
– phosphorylation of tyrosine kinases;
– activation of MAPKs;
– activation of PLA2 and arachidonic acid
metabolism;
– activation of PLD; and
– modulation of cyclic nucleotides
Early Signaling
 Activation of Tyrosine Kinases
• Ang II stimulates phosphorylation of a tyrosine
residue of many vascular smooth muscle cell
proteins.
• These include the AT1 receptor itself, PLC-γ1 and
Src family kinases (activated within seconds), as
well as JAK and TYK, FAK, Pyk2 (proline-rich
tyrosine kinase),p130Cas (a Crk-associated
substrate), and phosphatidylinositol3-kinase
(PI3K), all of which are activated within minutes
 Activation of Tyrosine Kinases
• The JAK (Janus Kinase)-STAT (signal
transducers and activators of transcription)
signaling pathway activates early growth
response genes and may be a mechanism
whereby Ang II influences vascular and cardiac
growth, remodeling, and repair
 Focal adhesion kinase and proline-rich
tyrosine kinase 2
• Ang II promotes cell migration and induces changes in
cell shape and volume by activating FAK dependent
signaling pathways
• Focal adhesion complexes, specialized sites of cell
adhesion, act as supramolecular structures for the
assembly of signal transduction mediators.
• The best characterized tyrosine kinase localized to focal
adhesion complexes is a 125-kDa protein, FAK.
• FAK is autophosphorylated at Tyr397 in resting
substrate-attached cells, and it possesses sites favored
for phosphorylation by Src
 FAK and PYK2
• FAK associates with paxillin and talin, and both
FAK and paxillin can bind to the cytoplasmic
tail of integrins independently
• Integrins are transmembrane receptors that
mediate the attachment between a cell and its
surroundings, such as other cells or the
extracellular matrix (ECM).
 Integrins
• Integrins pass information about the chemical
composition and mechanical status of the
ECM into the cell.
• In addition to transmitting mechanical forces
across otherwise vulnerable membranes, they
are involved in cell signaling and the
regulation of cell cycle, shape, and motility.
 FAK and PYK2
• FAK is abundant in developing blood vessels, and
elevation of its phosphotyrosine content in vascular
smooth muscle cells is a rapid response to Ang II
• Ang II-induced activation of FAK causes its
translocation to sites of focal adhesion with the
extracellular matrix and phosphorylation of paxillin and
talin, which may be involved in the regulation of cell
morphology and movement.
• The link between the AT1 receptor and FAK is
unknown, but the Rho family of GTPases are potential
candidates
 CADTK
• A novel p125FAK protein, calcium-dependent
tyrosine kinase (CADTK), has recently been
detected in rat aortic smooth muscle cells.
• CADTK is the rat homolog of Pyk2
• This tyrosine kinase is rapidly tyrosine-
phosphorylated by Ang II, and appears to be
associated with the cytoskeleton
 CADTK
• CADTK and FAK exhibit different modes of
activation.
• Activation of CADTK is highly correlated with the
stimulation of c-Jun N-terminal kinase (JNK)
activity, rather than with ERK activity, as is the
case for FAK
• In endothelial cells the balance of Pyk2 tyrosine
phosphorylation in response to Ang II is
controlled by Yes kinase and by a tyrosine
phosphatase SHP-2
 PYK2
• Pyk2: also called cell adhesion kinase-β
• FAK, PYK2 and CADTK are activated by GPCR
(AT1) and by PKC stimulation and increased
intracellular Ca2+
• The AT1 receptor uses Ca2+dependent PYK2 to
activate c-Src, required for Pyk2-mediatedERK
activation
 pyk2
• Since Pyk2 is a candidate to both regulate c-Src and
to link G protein coupled vasoconstrictor receptors
with protein tyrosine kinase-mediated contractile,
migratory, and growth responses, it may be a
potential point of convergence between
Ca2+dependent signaling pathways and protein
tyrosine kinase pathways in vascular smooth muscle
cells
 p130Cas
• p130Cas is an Ang II-activated tyrosine kinase
that plays a role in cytoskeletal rearrangement.
• This protein serves as an adapter molecule
because it contains proline-rich domains, an SH3
domain, and binding motifs for the SH2 domains
of Crk and Src
• p130Cas is important for integrin-mediated cell
adhesion, by recruitment of cytoskeletal signaling
molecules such as FAK, paxillin, and tensin to the
focal adhesions
 p130Cas
• Phosphorylation to be dependent on Ca2+, c-
Src, and PKC, and that it requires an intact
cytoskeletal network
• p130Cas has recently been demonstrated to
play a critical role in cardiovascular
development and actin filament assembly.
• Mice lacking p130Cas died in utero showing
marked venous congestion and growth
retardation.
 p130Cas
• Histologically, the heart was poorly developed
and blood vessels were prominently dilated
• p130Cas plays an essential role in arterial and
cardiac development, and accordingly in
remodeling in cardiovascular disease.
 Phosphatidylinositol 3-kinase (PI3-K)
• PI3Ks, a large family of intracellular signal
transducers that phosphorylate inositol lipids
at the 39 position of the inositol ring to
generate the 3-phosphoinositides PI(3)P,
PI(3,4)P2 and PI(3,4,5)P3, are heterodimeric
proteins composed of 85-and 110-kDa
subunits
 PI3K
• These kinases influence cell survival,
metabolism, cytoskeletal, reorganization, and
membrane trafficking and have recently been
identified to play an important role in the
regulation of vascular smooth muscle cell
growth
• PI3K, characteristically associated with
tyrosine kinase receptors, is also activated by
AT1 receptors
 PI3K
• Ang II stimulates activity, phosphorylation, and
migration of PI3K, and induces translocation of
the p85 subunit
• The action of Ang II peaks at 15 min and returns
to control levels by 30 min.
• PI3K inhibition by wortmannin and LY294002
completely blocks Ang II-stimulated hyperplasia
in cultured rat cells, suggesting the important
regulatory role in vascular smooth muscle cell
growth
 PI3K
• Several molecular targets for PI3K have been
identified, including centaurin, the actin-
binding protein profilin, phosphoinositide-
dependent kinases, the atypical PKCs, PLCγ,
Rac1, and JNK and the protein Ser/Thr kinase
Akt/protein kinaseB (PKB)
• Akt/PKB has recently been identified as an
important PI3K downstream target in Ang II-
activated VSMCs
 PI3K
• It regulates protein synthesis by activating p70
S6-kinase (p70S6K), and it modulates Ang II-
mediated Ca2+ responses in aortic cells by
stimulating Ca2+ channel currents.
• Akt/PKB has also been implicated to protect
vascular smooth muscle cells from apoptosis
and to promote cell survival by influencing
Bcl-2 and c-Myc expression and by inhibiting
caspases
Tyrosine kinase pathways stimulated by Ang II in
vascular smooth muscle cells
 Activation of Tyrosine Kinases

• Ang II rapidly activates Src, which regulates

PLC-γ- and ERK-dependent signaling pathways.
• Ang II binding to the AT1 receptor induces the
physical association and activation of
JAK2/TYK2 (Janus kinases).
• JAK2/TYK2 phosphorylates STAT proteins that
are translocated to the nucleus where they
activate gene transcription.
 Activation of Tyrosine Kinases
• Ang II also activates
– FAK, which possesses sites favored for phosphorylation by
Src.
– Pyk2 and CADTK are activated by Ang II through Ca2+-
dependent pathways.
– p130cas is transiently activated by Ang II, possibly via a
Ca2+-dependent pathway. Phosphorylated p130cas may be
important in the regulation of a-actin expression.
• PI3K activation by Ang II leads to Akt/PKB activation, which in
turn stimulates cell survival pathways and activation of
p70S6K. p70S6K, p70 S6-kinase.
 MAPK Pathways
• MAP kinases constitute a superfamily of
serine/threonine protein kinases involved in the
regulation of a number of intracellular pathways.
• Mammalian MAPKs are grouped into six major
subfamilies:
– ERK-1/ERK-2;
– JNK/stress-activated protein kinases (SAPK);
– p38;
– ERK-6, p38-like MAPK;
– ERK-3; and
– ERK-5 (also called Big MAP kinase 1)
Schematic diagram of the currently known
mammalian MAP kinase signaling pathways
 MAPK activation
• MAP kinase-dependent signaling pathways have
been associated with cellular growth and
apoptosis, with cellular differentiation and
transformation and with vascular contraction
• The ERKs are activated in response to growth and
differentiation factors, whereas JNKs and p38 are
usually activated in response to inflammatory
cytokines and cellular stress
• Ang II activates the three major members of the
MAP kinase family, ERKs, JNKs, and p38
 Activation of MAPK
• MAP kinase pathways comprise a three-
component protein kinase cascade consisting of a
serine/threonine protein kinase (MAPKKK), which
phosphorylates and activates a dual-specificity
protein kinase (MAPKK), which in turn
phosphorylates and activates another protein
kinase (MAPK).
• In the Ras/Raf/MEK/ ERK pathway, Raf
corresponds to MAPKKK, MEK corresponds to
MAPKK, and ERK corresponds to MAPK
 General Schema for MAPK Cascade
* ERK=Extracellular Signal-Regulated Kinase
* JNK=c-Jun N-terminal Kinase
* p38

Growth Factors Cytokines

Stimulus Cellular Stress
ROS / RNS
(Osmotic stress, ROS)
MAPK Kinase Kinase Raf
MEKK
MAPK Kinase MEK1/2
MEK3 MEK4
MAPK ERK1/2* p38 * JNK1/2*

Response Mitogenic Stress Responses

growth, differentiation
 Upstream events
Ang II-binding to AT1 receptors that induces
Shc-Grb2-Sos formation (tyrosine
phosphorylation of Shc), which in turn facilitates
guanine nucleotide exchange on the small G
protein Ras-GDP/GTP.

Activated Ras-GTP interacts with the Ser/Thr

kinase Raf (MAPK kinase kinase (MAPKKK)) which
translocates to the cell membrane.

Activation of Raf leads to phosphorylation of two

serine residues present in MEK (MAPK/ERK
kinase), which in turn phosphorylates Thr/Tyr and
activates MAPK, present as a 44- (ERK-1) and a
42-kDa (ERK-2) isoform.

Phosphorylated ERK has diverse intracellular

protein targets, which it phosphorylates
and activates

Dephosphorylation of ERK is accomplished by

activation of MAP kinase phosphatase-1 (MKP-1).
Downstream events
These substrates include:
1) proteins involved in transcriptional
activation such as Elk-1, TAL 1, RNA
polymerase II;
2) proteins involved in protein translation
such as PHAS-I;
3) structural proteins such as myelin
basic protein (MBP), microtubule
associated protein (MAP) and
caldesmon; and
4) secondary enzymes such as PLA2, S6
kinase, Ca2+ channels, Na+/H+ exchanger,
and MAP kinase activated
protein kinase (MAPKAPK2).

Activation of these downstream

proteins regulates cellular functions
associated with cell growth and
contraction
 MAPK
• Events downstream to MAP kinase activation are
numerous and heterogeneous and include PLA2,
cytoskeletal proteins, the MAPK-activated protein
kinase 2 (MAPKAPK-2), and the p90rsk protein
kinase, which can move to the nucleus and
activate transcription factors
• Once phosphorylated ERKs translocate to the
nucleus to phosphorylate transcription factors
and thereby regulate gene expression of cell
cycle-related proteins
 MAPK
• Both ERK-1/ERK-2 and JNK/SAPK lead to ternary
complex formation at the serum response element that
is present on many gene promoters, and to increased
transcriptional activity
• Alternatively, phosphorylation of the translation
regulator protein, PHAS-I (phosphorylated heat- and
acid-stable protein) promotes the dissociation of the
PHAS-I-eukaryotic initiation factor (eIF)-4E complex,
normally tightly bound when PHAS-I is relatively
underphosphorylated, releasing eIF-4E that will
facilitate initiation of translation in the nucleus
 P38 and diabetic kidney
• Inflammation and fibrosis are common disease
mechanisms involved in many forms of progressive
renal injury, including diabetic nephropathy
• P38 phosphorylation plays imp role in this process
• Specific inhibition of p38 MAPK signalling is
effective in the treatment of human endotoxaemia
(presence of toxin in blood), and in rat models of
inflammatory renal disease, lung fibrosis and
arthritis
 p38
• In vitro studies have shown that components
of the diabetic milieu (high glucose and
advanced glycation end-products) can activate
p38 MAPK signalling in renal cells.
• Exposure to high glucose levels induces p38
MAPK phosphorylation in mesangial cells and
glycated albumin stimulates p38 MAPK
phosphorylation in fibroblasts
 p38
• The diabetic milieu may also promote p38
MAPK phosphorylation in diabetic kidneys by
inducing inflammation and cytokine
production
 Apoptosis
 Is a highly organized orchestrated form of cell death ( cellular suicide;
programmed cell death)
 Apoptosis can be triggered by stress – oxidants (ROS), DNA damage agents (UV,
irradiation), chemotherapeutic drugs
 Morphological Changes
– Withered and shrunken appearance
– Blebbing of the cell membrane
– Cell no longer adheres to neighbors
 Biochemical Changes
– Proteolysis of the cytoskeleton
– Proteolysis of structural proteins supporting the nuclear membrane
– Degradation of chromosomal DNA (Characteristic ladder pattern on agarose
gel)
– Cell contents eaten by neighbors
 The Players

Regulators
Executioners
 Apoptosis Activators
 Several Proteases
Cell surface proteins (Fas ligand, Fas)
(The caspases)
 Adaptor proteins/domains
Fas associated death domains (FADD)
 One Nuclease
Caspase recruitment domains (CARD)
(Caspase-activated DNase)
 Apoptotic protease activating factor
(Apaf-1)
 Cytochrome c

Inhibitors of Apoptosis

 The Bcl-2/Bax family

 Caspases: Executioners

• Cysteine proteases that cleave at aspartic acid residues

• Highly specific: each has its target protein

• Caspases are constitutively expressed in inactive zymogen

form as procaspases

• Activated by specific proteolysis- often by another caspase.

H2O2 induced activation of ERK, p38 and Akt in
a time dependent manner
• RPTC were
exposed to 1
mM H2O2 for
the indicated
time periods.
Cell lysates
were separated
by SDS-PAGE
and
immunoblotted
with antibodies
to phosphor
(p)-ERK1/2,
ERK1/2,
phospho-Akt,
Akt, phospho-
p38, and p38.
Activation of Phospholipase A2 and Arachidonic
Acid Metabolism
• Ang II stimulates PLA2 activity, which is
responsible for the release of arachidonic acid
from cell membrane phospholipids
• Released AA will be processed by
cyclooxygenases, lipoxygenases, or
cytochrome P450 oxygenases to many
different eicosanoids in vascular and renal
tissues
 PLA2
• PLA2-derived eicosanoids influence vascular
and renal mechanisms important in blood
pressure regulation
• Vascular PLA2 activity in response to Ang II is
evident within minutes and is sustained for at
least 30 min after Ang II stimulation
• In vascular smooth muscle cells and
endothelial cells, these effects are mediated
via AT1 receptors
 PLA2
• In neonatal rat cardiac myocytes, neuronal cells, and renal
proximal tubule epithelial cells, Ang II-induced activation of
PLA2 occurs via AT2 receptors
• Ang II-elicited activation of vascular PLA2 is dependent on
[Ca2+]i, Ca2+-calmodulin-dependent protein kinase II (CaM
kinase II), and MAP kinases.
• Activated PLA2 and its metabolites in turn activate Ras/MAP
kinase-dependent signaling pathways, amplifying PLA2
activity and releasing additional arachidonic acid by a
positive feedback mechanism
• Ang II-generated eicosanoids regulate vascular contraction
and growth, possibly by activating MAP kinases
 Phospholipase D activation
• PLD, which hydrolyzes phospholipids (mainly
phosphatidylcholine) to generate phosphatidic
acid, is a critical component in cellular
signaling associated with mitogenesis
• Sustained activation of PLD is a major source
of prolonged second messenger generation in
vascular smooth muscle cells and
cardiomyocytes.
 PLD
• Unlike PLC, which is activated within seconds by Ang II,
PLD activation is detectable at about 2 min and remains
elevated for up to 60 min
• The downstream pathways associated with Ang II-
induced activation of PLD in vascular smooth muscle
cells are PKC-independent but involve intracellular Ca2+
mobilization and Ca2+ influx that is tyrosine kinase-
dependent
• Ang II-induced PLD signaling has been implicated in
cardiac hypertrophy as well as in proliferation of
vascular smooth muscle cells
Angiotensin II Effects on Cyclic Nucleotides

• The cyclic nucleotides cAMP and cGMP are

generated intracellularly within minutes by
adenylate cyclase and guanylate cyclase
• Downstream targets of cyclic nucleotides
include cAMP-dependent protein kinase,
cGMP-dependent protein kinase,intracellular
Ca2+, and ionic channels
Angiotensin II Effects on Cyclic Nucleotides

• Increased cyclic nucleotide concentration leads to

decreased [Ca2+]i and reduced Ca2+ sensitivity of
phosphorylation in vascular smooth muscle, with
resultant smooth muscle relaxation.
• Ang II influences vascular dilation either directly,
by increasing intracellular cAMP and cGMP
concentrations, or indirectly, by potentiating
vasodilator-induced formation of cyclic
nucleotides.
 Early Signaling

• These processes are initiated by signaling

pathways that are stimulated by Ang II within
minutes and include:
– phosphorylation of tyrosine kinases;
– activation of MAPKs;
– activation of PLA2 and arachidonic acid
metabolism;
– activation of PLD; and
– modulation of cyclic nucleotides
 Long-Term Effects Mediated by Angiotensin II

• Ang II influences the long-term control of

cellular growth, adhesion, and migration, as
well as intercellular matrix deposition within
the vasculature and the heart thereby
influencing chronic adaptive changes in
vascular remodeling, cardiac hypertrophy, as
well as processes involved in atherosclerosis.
 Long term effects of Ang II
• Intracellular cascades underlying long-term Ang II
signaling involve
– early activation of various kinases
– that phosphorylate downstream targets regulating
chronic and sustained cellular functions.
– Stimulation of redox-sensitive pathways, induction of
proto-oncogene expression,
– cross-talk with tyrosine kinase receptors,
– production of other growth factors and stimulation of
nuclear signaling cascades ultimately result in cellular
growth and differentiaition
Long-term signaling events
Generation of reactive oxygen species
• Reactive oxygen species such as superoxide
anions and hydrogen peroxide act as
intercellular and intracellular second
messengers that may play a physiological role
in vascular tone and cell growth, and a
pathophysiological role in inflammation,
ischemia-reperfusion, hypertension, and
atherosclerosis
Generation of reactive oxygen species
Generation of reactive oxygen species
• Xanthine oxidase, mitochondrial oxidases and
arachidonic acid are the major sources of
oxidative molecules in nonvascular tissue
whereas a non mitochondrial, membrane
associated NADH/NADPH oxidase appears to be
the most important source of superoxide anion in
vascular cells
• This enzyme transfers electrons from NADH or
NADPH to molecular oxygen, producing
superoxide anion
Generation of reactive oxygen species
• The complete molecular structure of the vascular
oxidase is unknown, but it shares some features
with the neutrophil oxidase.
• In neutrophils, NADH/NADPH oxidase consists of
five subunits:
– a 22-kDa a-subunit(p22phox),
– a glycosylated 91-kDa b-subunit (gp91phox),which
together make up cytochrome b558, the electron
transfer element;
– cytosolic components p47phox and p67phox;
– and a low-molecular weight G protein, rac1 or rac2
Generation of reactive oxygen species
• Upon activation, the p47phox and p67phox proteins
are translocated to the membrane and associate
with the cytochrome b558, creating the active
oxidase.
• In vascular smooth muscle cells, p22phox is a
critical component of the superoxide-generating
NADH/NADPH oxidase system
• Ang II activation of NADH/NADPH oxidase is
delayed and is only detectable in vascular smooth
muscle cells about 60min after Ang II stimulation
Generation of reactive oxygen species
• Generation of reactive oxygen species is
regulated by various cytokines and growth
factors, including Ang II, which increases O2. and
H2O2 production in cardiac,vascular smooth
muscle, endothelial, adventitial, and mesangial
cells
• and generation of reactive oxygen species has
been implicated in the pathogenesis of Ang II-
induced but not catecholamine-induced
hypertension
Generation of reactive oxygen species
• Mechanisms underlying oxidative stress-
induced hypertension may be associated with
degradation of endothelium derived NO and
with the potent vascular mitogenic effects of
O2 . and H2O2
• Growth of vascular smooth muscle cells has
an essential redox-sensitive component,
which appears to be mediated in part via
activation of ERK-5
Generation of reactive oxygen species
• Reactive oxygen species stimulate hyperplasia
and hypertrophy of vascular smooth muscle
cells, whereas antioxidants inhibit growth,
trigger apoptosis, and attenuate the response
to growth factors and hypertrophic agents
• Stimulate endothelial vascular cell adhesion
molecule-1, important in cell-cell interactions,
and possibly in processes associated with
atherosclerosis
 Tempol
• Tempol (membrane permeable SOD mimetic)
was reported to reduce the blood pressure in
zinc, or dexamethasone, or DOCA salt or L-
NAME-induced hypertension and in Dahl
sensitive rats
• Tempol restored the augmented Ang II-
mediated responses in SHR, STZ diabetic rats
and high glucose concentration in vitro
HFD and Oxidative stress
Oxidative stress and Blood Pressure
Tempol and Ang II
Oxidative stress and Endothelium
 Tempol
• Tempol attenuates the Ang II-mediated
enhanced responses in HFD-fed rats in dose-
dependent manner, thus supporting the
superoxide hypothesis in enhanced response
to Ang II in insulin resistance.
• Tempol also improves the endothelial
dysfunction in STZ diabetic rats
• SOD mimetic tempol reversed vascular
dysfunction by scavenging superoxides
 Expression of Proto-Oncogenes and
Growth Factors
• Long-term control of Ang II-regulated cellular growth,
adhesion, migration, fibrosis, and collagen deposition
within the vasculature involves protein synthesis
• Ang II induces the expression of several proto-
oncogenes in human and rat vascular smooth muscle
cells, including c-fos, c-jun, cmyc, erg-1, VL-30, proto-
oncogene/activator protein 1 complex
• Ang II increases expression of vascular c-fos in a PKC
and Ca2+-dependent manner via multiple regulatory
mechanisms
 Expression of Proto-Oncogenes
• The c-fos promoter contains a cAMP/calcium
response element (CRE), a serum response
element (SRE), and a sis-inducing factor element
(SIE)
• These promoter elements are regulated by
various proteins activated by Ang II, including
cAMP and PKA, which regulate CRE, MAPK-
stimulated phosphorylation of p62TCF and PKC,
which regulate SRE, and STATs, which regulate SIE
 Growth factors
• Stimulation of early response genes by Ang II is
associated with increased gene expression and
production of
– growth factors, such as PDGF, EGF, transforming growth
factor-b (TGF-b), insulin-like growth factor-1 (IGF-1), basic
fibroblast growth factor (bFGF) and platelet activating
factor (PAF)
– vasoconstrictor agents, such as ET-1,
– adhesion molecules such as ICAM-1, VCAM-1, and E-
selectin, and integrins
– chemotactic factors such as tumor necrosis factor-α (TNF-
α) and monocyte chemoattractant protein-1 (MCP-1)
Altered Angiotensin II Signaling and Hypertension

• Alterations in some of
the short-term Ang II-
stimulated signaling
events in
hypertension.
• In hypertension, PLC
activity, IP3
generation, and DAG
production are
increased in response
to Ang II stimulation.
Alterations in some
of the long-term
Ang II-stimulated
signaling events in
hypertension
 Ang II and Hypertension
• Ang II influences arterial tone and remodeling
in hypertension by stimulating vascular
smooth muscle cell contraction, augmenting
cell growth, increasing deposition of
extracellular matrix, inhibiting apoptosis,
inducing cell migration, and promoting
inflammation
 Ang II and Hypertension
• Recent data showing that ACE inhibitors or
AT1 receptor antagonist treatment of
essential hypertensive patients regresses
structural and functional abnormalities of the
vascular wall suggest that Ang II plays a critical
role in abnormal behavior of vascular cells in
hypertension.
 Ang II and Hypertension
• Mechanisms underlying these cellular effects
seem to occur at the post-receptor level and
appear to be associated with
– hyperactivity of Ang II-stimulated G protein-
coupled phospholipases, tyrosine kinase-, and
MAP kinase-dependent pathways, as well as with
oxidative stress