• ECs :

  • receive and translate signals from the blood

  • Barrier

  • Blood flow - via interaction with VSMC

  • Hemostasis ( stop bleeding )

  • Anti-coagulant

  • Angiogenesis

  • Inflammatory response - extravasation of leukocytes into tissues

• Intracellular calcium increases in response to G protein coupled receptor binding proinflammatory mediators such as chemokines, activated complement

• Low pH and lactate induce massive amounts of intracellular calcium release dysregulate vascular tone and lead to EC apoptosis

• Calcium Mediates Release of Various Relaxing Factors from Endothelial Cells

Know the layers of blood vessels , their cellular composition , and difference between arteries , veins , and capillaries

Layers of blood vessels :

• Tunica intima : Endothelial cells + basement membrane

• Tunica media : Primarily smooth muscle cells ( VSMCs )

• Tunica externa ( adventitia ) : Fibroblasts , vaso vasorum , extracellular matrix

Differences :

• Arteries :

  • Thick tunica media

  • Internal and external elastic lamina ( IEL / EEL ) present

• Veins :

  • Thinner tunica media

  • No IEL or EEL

  • Thicker tunica externa

• Capillaries :

  • Single layer of endothelial cells

  • Three types : continuous , fenestrated , and sinusoidal ( based on permeability and location : e.g., adipose , intestine , spleen )

Describe the functions of smooth muscle cells and endothelial cells

• Act as a barrier

• Regulate blood flow and vascular tone via signaling with VSMCs

• Maintain hemostasis :

  • Anti-coagulant surface

• Mediate angiogenesis ( formation of new blood vessels )

• Facilitate leukocyte trafficking and respond to inflammation

Smooth muscle cells ( VSMCs ) :

• Maintain vascular tone and blood pressure via contraction and relaxation

• Can undergo a phenotypic switch from quiescent ( contractile ) to proliferative ( synthetic ) in response to injury or disease

• Regulated by intracellular calcium , NO ( nitric oxide ) , and calmodulin

Understand the various ways that endothelial cells and smooth muscle cells communicate and their implications

Mechanisms of EC-VSMC communication :

1. Direct contact :

   • Myoendothelial junctions ( MEJs ) : Transfer ions ( Ca²⁺ ) , second messengers ( IP₃ , cAMP )

   • Notch signaling ( e.g., Jagged1 / Notch3 )

   • Ephrin / Eph interactions

2. Nitric Oxide ( NO ) from ECs :

   • Activates myosin light chain phosphatase → VSMC relaxation

   • Anti-atherosclerotic effects : inhibits VSMC proliferation , reduces platelet aggregation , and leukocyte adhesion

3. Extracellular matrix ( ECM ) :

   • EC glycocalyx and perlecan ( HSPG ) regulate cytokine binding ( e.g., IL-2-induced VSMC proliferation )

4. miRNAs :

   • EC-derived miR-92a modulates VSMC phenotype , increases blood pressure , reduces NO levels

5. Extracellular vesicles :

   • Exosomes ( 30–150 nm ) , microvesicles ( 100–1000 nm ) , apoptotic bodies

   • Carry miRNAs , proteins , and signaling molecules

Implications :

• Dysregulation leads to atherosclerosis , hypertension , vascular stiffness , and endothelial dysfunction

Describe different means by which endothelial and smooth muscle cell interactions can be studied in vitro

Indirect co-culture :

• Transwell ( Boyden chamber ) : ECs and VSMCs are cultured separately but share soluble factors

• Conditioned media : Media from ECs applied to VSMCs ( or vice versa )

• Isolated extracellular vesicles : Applied to target cells to study paracrine effects

Direct co-culture :

• Matrix-like gels : ECs and VSMCs co-seeded on substrates mimicking ECM

• Spheroids : 3D aggregates showing enhanced tissue-specific behavior , exosome production

• Organoids : Derived from pluripotent stem cells

• Vessels-on-a-chip : Microfluidic devices using ECs and VSMCs to mimic vasculature

Paper Discussion

• VSM and EC cells are required for vascular homeostasis

• calmodulin = required for VSMc function

• Unknown = if EC's regulate vascular functions

• Background = CAM interacts with over 300 cellular proteins

  • smooth muscle cells have about 5% availability of CAM

• Rationale = increase CAM in VSMs

• Gap in Knowledge = VCs use CAM to regulate function

• Methods = Ca Sensor Protein = calmodulin

• Endothelial Cells Secrete Soluble Factors

• eNOS has high affinity for calmodulin

• 20% EC with 70% VSMc co-culture = greatest amount of calmodulin expression

• Negative FRET analysis = used to find degree of calmodulin present

• Greater amount of comodulin in co-cultured cells ,

  • no difference in calcium levels

• Calmodulin mRNA and protein is upregulated in VSMs co-cultured with 20% ECs

• Some unknown factor regulates calmodulin levels

• VEGF = enhances calmodulin expression

• Block VEGF = blocks angiogenesis in tumor formation

• VEGF uses IP3 to promote calmodulin expression

• Can't be contact based signal , must be soluble diffusion factor